Coronavirus vaccine

ABSTRACT

This disclosure relates to the field of RNA to prevent or treat coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses such as antibody and/or T cell responses. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccine antigen.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirely. Said ASCII copy, created on Mar. 17, 2022, isnamed 2013237-0305_SL.txt and is 169,810 bytes in size.

This disclosure relates to the field of RNA to prevent or treatcoronavirus infection. In particular, the present disclosure relates tomethods and agents for vaccination against coronavirus infection andinducing effective coronavirus antigen-specific immune responses such asantibody and/or T cell responses. These methods and agents are, inparticular, useful for the prevention or treatment of coronavirusinfection. Administration of RNA disclosed herein to a subject canprotect the subject against coronavirus infection. Specifically, in oneembodiment, the present disclosure relates to methods comprisingadministering to a subject RNA encoding a peptide or protein comprisingan epitope of SARS-CoV-2 spike protein (S protein) for inducing animmune response against coronavirus S protein, in particular S proteinof SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccineantigen. Administering to the subject RNA encoding vaccine antigen mayprovide (following expression of the RNA by appropriate target cells)vaccine antigen for inducing an immune response against vaccine antigen(and disease-associated antigen) in the subject.

In December 2019, a pneumonia outbreak of unknown cause occurred inWuhan, China and it became clear that a novel coronavirus (severe acuterespiratory syndrome coronavirus 2; SARS-CoV-2) was the underlyingcause. The genetic sequence of SARS-CoV-2 became available to the WHOand public (MN908947.3) and the virus was categorized into thebetacoronavirus subfamily. By sequence analysis, the phylogenetic treerevealed a closer relationship to severe acute respiratory syndrome(SARS) virus isolates than to another coronavirus infecting humans,namely the Middle East respiratory syndrome (MERS) virus. On February2nd, a total of 14,557 cases were globally confirmed in 24 countriesincluding Germany and a subsequent self-sustaining, human-to-human virusspread resulted in that SARS-CoV-2 became a global epidemic.

Coronaviruses are positive-sense, single-stranded RNA ((+)ssRNA)enveloped viruses that encode for a total of four structural proteins,spike protein (S), envelope protein (E), membrane protein (M) andnucleocapsid protein (N). The spike protein (S protein) is responsiblefor receptor-recognition, attachment to the cell, infection via theendosomal pathway, and the genomic release driven by fusion of viral andendosomal membranes. Though sequences between the different familymembers vary, there are conserved regions and motifs within the Sprotein making it possible to divide the S protein into two subdomains:S1 and S2. While the S2, with its transmembrane domain, is responsiblefor membrane fusion, the S1 domain recognizes the virus-specificreceptor and binds to the target host cell. Within several coronavirusisolates, the receptor binding domain (RBD) was identified and a generalstructure of the S protein defined (FIG. 1 ).

Vaccine approaches and therapeutics against SARS-CoV-2 are currently notavailable, but urgently needed.

Due to the importance of the S protein in host cell recognition andentry, as well as in the induction of virus neutralising antibodies bythe host immune system, we decided to target the viral S protein ofSARS-CoV-2 and subdomains of the S protein such as S1 or RBD for vaccinedevelopment. Mutations within the regions important for conformationmight be beneficial for inducing a stronger protective immune response.Therefore, we envision testing several constructs (FIG. 2 ). As thenaïve S protein is a trimer and this trimeric structure has most likelyan effect on the stability of the protein and the antigenicity, weincluded a strategy based on a stabilized construct introducing the T4bacteriophage fibritin domain which is also in use in HIV for generatingstable gp140 trimers and functional for SARS RBD-constructs.

SUMMARY

The present invention generally embraces the immunotherapeutic treatmentof a subject comprising the administration of RNA, i.e., vaccine RNA,encoding an amino acid sequence, i.e., a vaccine antigen, comprisingSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof,i.e., an antigenic peptide or protein. Thus, the vaccine antigencomprises an epitope of SARS-CoV-2 S protein for inducing an immuneresponse against coronavirus S protein, in particular SARS-CoV-2 Sprotein, in the subject. RNA encoding vaccine antigen is administered toprovide (following expression of the polynucleotide by appropriatetarget cells) antigen for induction, i.e., stimulation, priming and/orexpansion, of an immune response, e.g., antibodies and/or immuneeffector cells, which is targeted to target antigen (coronavirus Sprotein, in particular SARS-CoV-2 S protein) or a procession productthereof. In one embodiment, the immune response which is to be inducedaccording to the present disclosure is a B cell-mediated immuneresponse, i.e., an antibody-mediated immune response. Additionally oralternatively, in one embodiment, the immune response which is to beinduced according to the present disclosure is a T cell-mediated immuneresponse. In one embodiment, the immune response is an anti-coronavirus,in particular anti-SARS-CoV-2 immune response.

The vaccine described herein comprises as the active principlesingle-stranded RNA that may be translated into the respective proteinupon entering cells of a recipient. In addition to wildtype orcodon-optimized sequences encoding the antigen sequence, the RNA maycontain one or more structural elements optimized for maximal efficacyof the RNA with respect to stability and translational efficiency (5′cap, 5′ UTR, 3′ UTR, poly(A)-tail). In one embodiment, the RNA containsall of these elements. In one embodiment, beta-S-ARCA(D1) (m₂^(7,2′-O)GppSpG) or m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG may be utilized asspecific capping structure at the 5′-end of the RNA drug substances. As5′-UTR sequence, the 5′-UTR sequence of the human alpha-globin mRNA,optionally with an optimized ‘Kozak sequence’ to increase translationalefficiency may be used. As 3′-UTR sequence, a combination of twosequence elements (FI element) derived from the “amino terminal enhancerof split” (AES) mRNA (called F) and the mitochondrial encoded 12Sribosomal RNA (called I) placed between the coding sequence and thepoly(A)-tail to assure higher maximum protein levels and prolongedpersistence of the mRNA may be used. These were identified by an ex vivoselection process for sequences that confer RNA stability and augmenttotal protein expression (see WO 2017/060314, herein incorporated byreference). Alternatively, the 3′-UTR may be two re-iterated 3′-UTRs ofthe human beta-globin mRNA. Furthermore, a poly(A)-tail measuring 110nucleotides in length, consisting of a stretch of 30 adenosine residues,followed by a 10 nucleotide linker sequence (of random nucleotides) andanother 70 adenosine residues may be used. This poly(A)-tail sequencewas designed to enhance RNA stability and translational efficiency.

Furthermore, a secretory signal peptide (sec) may be fused to theantigen-encoding regions preferably in a way that the sec is translatedas N terminal tag. In one embodiment, sec corresponds to the secreotorysignal peptide of the S protein. Sequences coding for short linkerpeptides predominantly consisting of the amino acids glycine (G) andserine (S), as commonly used for fusion proteins may be used asGS/Linkers.

The vaccine RNA described herein may be complexed with proteins and/orlipids, preferably lipids, to generate RNA-particles for administration.If a combination of different RNAs is used, the RNAs may be complexedtogether or complexed separately with proteins and/or lipids to generateRNA-particles for administration.

In one aspect, the invention relates to a composition or medicalpreparation comprising RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

In one embodiment, an immunogenic fragment of the SARS-CoV-2 S proteincomprises the S1 subunit of the SARS-CoV-2 S protein, or the receptorbinding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is able toform a multimeric complex, in particular a trimeric complex. To thisend, the amino acid sequence comprising a SARS-CoV-2 S protein, animmunogenic variant thereof, or an immunogenic fragment of theSARS-CoV-2 S protein or the immunogenic variant thereof may comprise adomain allowing the formation of a multimeric complex, in particular atrimeric complex of the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof. In oneembodiment, the domain allowing the formation of a multimeric complexcomprises a trimerization domain, for example, a trimerization domain asdescribed herein.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is encodedby a coding sequence which is codon-optimized and/or the G/C content ofwhich is increased compared to wild type coding sequence, wherein thecodon-optimization and/or the increase in the G/C content preferablydoes not change the sequence of the encoded amino acid sequence.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment ofthe amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 ofSEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 17 to685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of theamino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 17 to 685 ofSEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 ofSEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 17 to1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenicfragment of the amino acid sequence of amino acids 17 to 1273 of SEQ IDNO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof comprises asecretory signal peptide.

In one embodiment, the secretory signal peptide is fused, preferablyN-terminally, to a SARS-CoV-2 S protein, an immunogenic variant thereof,or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof.

In one embodiment,

(i) the RNA encoding the secretory signal peptide comprises thenucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9;and/or(ii) the secretory signal peptide comprises the amino acid sequence ofamino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to16 of SEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence of SEQ IDNO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, ora fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of SEQ ID NO: 6; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of SEQ ID NO: 5, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 5, or animmunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 5.

In one embodiment, the RNA is a modified RNA, in particular a stabilizedmRNA. In one embodiment, the RNA comprises a modified nucleoside inplace of at least one uridine. In one embodiment, the RNA comprises amodified nucleoside in place of each uridine. In one embodiment, themodified nucleoside is independently selected from pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In one embodiment, the RNA comprises a modified nucleoside in place ofuridine.

In one embodiment, the modified nucleoside is selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U).

In one embodiment, the RNA comprises a 5′ cap.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 5′ UTR comprising the nucleotide sequence of SEQ ID NO: 12,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 3′ UTR comprising the nucleotide sequence of SEQ ID NO: 13,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a poly-A sequence.

In one embodiment, the poly-A sequence comprises at least 100nucleotides.

In one embodiment, the poly-A sequence comprises or consists of thenucleotide sequence of SEQ ID NO: 14.

In one embodiment, the RNA is formulated or is to be formulated as aliquid, a solid, or a combination thereof.

In one embodiment, the RNA is formulated or is to be formulated forinjection.

In one embodiment, the RNA is formulated or is to be formulated forintramuscular administration.

In one embodiment, the RNA is formulated or is to be formulated asparticles.

In one embodiment, the particles are lipid nanoparticles (LNP) orlipoplex (LPX) particles.

In one embodiment, the LNP particles comprise((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

In one embodiment, the RNA lipoplex particles are obtainable by mixingthe RNA with liposomes. In one embodiment, the RNA lipoplex particlesare obtainable by mixing the RNA with lipids.

In one embodiment, the RNA is formulated or is to be formulated ascolloid. In one embodiment, the RNA is formulated or is to be formulatedas particles, forming the dispersed phase of a colloid. In oneembodiment, 50% or more, 75% or more, or 85% or more of the RNA arepresent in the dispersed phase. In one embodiment, the RNA is formulatedor is to be formulated as particles comprising RNA and lipids. In oneembodiment, the particles are formed by exposing RNA, dissolved in anaqueous phase, with lipids, dissolved in an organic phase. In oneembodiment, the organic phase comprises ethanol. In one embodiment, theparticles are formed by exposing RNA, dissolved in an aqueous phase,with lipids, dispersed in an aqueous phase. In one embodiment, thelipids dispersed in an aqueous phase form liposomes.

In one embodiment, the RNA is mRNA or saRNA.

In one embodiment, the composition or medical preparation is apharmaceutical composition.

In one embodiment, the composition or medical preparation is a vaccine.

In one embodiment, the pharmaceutical composition further comprises oneor more pharmaceutically acceptable carriers, diluents and/orexcipients.

In one embodiment, the composition or medical preparation is a kit.

In one embodiment, the RNA and optionally the particle formingcomponents are in separate vials.

In one embodiment, the kit further comprises instructions for use of thecomposition or medical preparation for inducing an immune responseagainst coronavirus in a subject.

In one aspect, the invention relates to the composition or medicalpreparation described herein for pharmaceutical use.

In one embodiment, the pharmaceutical use comprises inducing an immuneresponse against coronavirus in a subject.

In one embodiment, the pharmaceutical use comprises a therapeutic orprophylactic treatment of a coronavirus infection.

In one embodiment, the composition or medical preparation describedherein is for administration to a human.

In one embodiment, the coronavirus is a betacoronavirus.

In one embodiment, the coronavirus is a sarbecovirus.

In one embodiment, the coronavirus is SARS-CoV-2.

In one aspect, the invention relates to a method of inducing an immuneresponse against coronavirus in a subject comprising administering tothe subject a composition comprising RNA encoding an amino acid sequencecomprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof.

In one embodiment, an immunogenic fragment of the SARS-CoV-2 S proteincomprises the S1 subunit of the SARS-CoV-2 S protein, or the receptorbinding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is able toform a multimeric complex, in particular a trimeric complex. To thisend, the amino acid sequence comprising a SARS-CoV-2 S protein, animmunogenic variant thereof, or an immunogenic fragment of theSARS-CoV-2 S protein or the immunogenic variant thereof may comprise adomain allowing the formation of a multimeric complex, in particular atrimeric complex of the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof. In oneembodiment, the domain allowing the formation of a multimeric complexcomprises a trimerization domain, for example, a trimerization domain asdescribed herein.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is encodedby a coding sequence which is codon-optimized and/or the G/C content ofwhich is increased compared to wild type coding sequence, wherein thecodon-optimization and/or the increase in the G/C content preferablydoes not change the sequence of the encoded amino acid sequence.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment ofthe amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 ofSEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 17 to685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of theamino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 17 to 685 ofSEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence ofnucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 ofSEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of amino acids 17 to1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenicfragment of the amino acid sequence of amino acids 17 to 1273 of SEQ IDNO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof comprises asecretory signal peptide.

In one embodiment, the secretory signal peptide is fused, preferablyN-terminally, to a SARS-CoV-2 S protein, an immunogenic variant thereof,or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof.

In one embodiment,

(i) the RNA encoding the secretory signal peptide comprises thenucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9;and/or(ii) the secretory signal peptide comprises the amino acid sequence ofamino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to16 of SEQ ID NO: 1.

In one embodiment,

(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof comprises the nucleotide sequence of SEQ IDNO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, ora fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of SEQ ID NO: 6; and/or(ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprises the amino acid sequence of SEQ ID NO: 5, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 5, or animmunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 5.

In one embodiment, the RNA is a modified RNA, in particular a stabilizedmRNA. In one embodiment, the RNA comprises a modified nucleoside inplace of at least one uridine. In one embodiment, the RNA comprises amodified nucleoside in place of each uridine. In one embodiment, themodified nucleoside is independently selected from pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In one embodiment, the RNA comprises a modified nucleoside in place ofuridine.

In one embodiment, the modified nucleoside is selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U).

In one embodiment, the RNA comprises a cap.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 5′ UTR comprising the nucleotide sequence of SEQ ID NO: 12,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 3′ UTR comprising the nucleotide sequence of SEQ ID NO: 13,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

In one embodiment, the RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a poly-A sequence.

In one embodiment, the poly-A sequence comprises at least 100nucleotides.

In one embodiment, the poly-A sequence comprises or consists of thenucleotide sequence of SEQ ID NO: 14.

In one embodiment, the RNA is formulated as a liquid, a solid, or acombination thereof.

In one embodiment, the RNA is administered by injection.

In one embodiment, the RNA is administered by intramuscularadministration.

In one embodiment, the RNA is formulated as particles.

In one embodiment, the particles are lipid nanoparticles (LNP) orlipoplex (LPX) particles.

In one embodiment, the LNP particles comprise((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

In one embodiment, the RNA lipoplex particles are obtainable by mixingthe RNA with liposomes. In one embodiment, the RNA lipoplex particlesare obtainable by mixing the RNA with lipids.

In one embodiment, the RNA is formulated as colloid. In one embodiment,the RNA is formulated as particles, forming the dispersed phase of acolloid. In one embodiment, 50% or more, 75% or more, or 85% or more ofthe RNA are present in the dispersed phase. In one embodiment, the RNAis formulated as particles comprising RNA and lipids. In one embodiment,the particles are formed by exposing RNA, dissolved in an aqueous phase,with lipids, dissolved in an organic phase. In one embodiment, theorganic phase comprises ethanol. In one embodiment, the particles areformed by exposing RNA, dissolved in an aqueous phase, with lipids,dispersed in an aqueous phase. In one embodiment, the lipids dispersedin an aqueous phase form liposomes.

In one embodiment, the RNA is mRNA or saRNA.

In one embodiment, the method is a method for vaccination againstcoronavirus.

In one embodiment, the method is a method for therapeutic orprophylactic treatment of a coronavirus infection.

In one embodiment, the subject is a human.

In one embodiment, the coronavirus is a betacoronavirus.

In one embodiment, the coronavirus is a sarbecovirus.

In one embodiment, the coronavirus is SARS-CoV-2.

In one embodiment of the method described herein, the composition is acomposition described herein.

In one aspect, the invention relates to a composition or medicalpreparation described herein for use in a method described herein.

Among other things, the present disclosure demonstrates that acomposition comprising a lipid nanoparticle encapsulated mRNA encodingat least a portion (e.g., that is or comprises an epitope) of aSARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein)can achieve detectable antibody titer against the epitope in serumwithin 7 days after administration to a population of adult humansubjects according to a regimen that includes administration of at leastone dose of the vaccine composition. Moreover, the present disclosuredemonstrates persistence of such antibody titer. In some embodiments,the present disclosure demonstrates increased such antibody titer when amodified mRNA is used, as compared with that achieved with acorresponding unmodified mRNA.

In some embodiments, a provided regimen includes at least one dose. Insome embodiments, a provided regimen includes a first dose and at leastone subsequent dose. In some embodiments, the first dose is the sameamount as at least one subsequent dose. In some embodiments, the firstdose is the same amount as all subsequent doses. In some embodiments,the first dose is a different amount as at least one subsequent dose. Insome embodiments, the first dose is a different amount than allsubsequent doses. In some embodiments, a provided regimen comprises twodoses. In some embodiments, a provided regimen consists of two doses.

In particular embodiments, the immunogenic composition is formulated asa single-dose in a container, e.g., a vial. In some embodiments, theimmunogenic composition is formulated as a multi-dose formulation in avial. In some embodiments, the multi-dose formulation includes at least2 doses per vial. In some embodiments, the multi-dose formulationincludes a total of 2-20 doses per vial, such as, for example, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, eachdose in the vial is equal in volume. In some embodiments, a first doseis a different volume than a subsequent dose.

A “stable” multi-dose formulation exhibits no unacceptable levels ofmicrobial growth, and substantially no or no breakdown or degradation ofthe active biological molecule component(s). As used herein, a “stable”immunogenic composition includes a formulation that remains capable ofeliciting a desired immunologic response when administered to a subject.

In some embodiments, the multi-dose formulation remains stable for aspecified time with multiple or repeated inoculations/insertions intothe multi-dose container. For example, in some embodiments themulti-dose formulation may be stable for at least three days with up toten usages, when contained within a multi-dose container. In someembodiments, the multi-dose formulations remain stable with 2-20inoculations/insertions.

In some embodiments, administration of a composition comprising a lipidnanoparticle encapsulated mRNA encoding at least a portion (e.g., thatis or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g.,of a SARS-CoV-2-encoded S protein), e.g., according to a regimen asdescribed herein, may result in lymphopenia in some subjects (e.g., inall subjects, in most subjects, in about 50% or fewer, in about 40% orfewer, in about 40% or fewer, in about 25% or fewer, in about 20% orfewer, in about 15% or fewer, in about 10% or fewer, in about 5% orfewer, etc). Among other things, the present disclosure demonstratesthat such lymphopenia can resolve over time. For example, in someembodiments, lymphopenia resolves within about 14, about 10, about 9,about 8, about 7 days or less. In some embodiments, lymphopenia is Grade3, Grade 2, or less.

Thus, among other things, the present disclosure provides compositionscomprising a lipid nanoparticle encapsulated mRNA encoding at least aportion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encodedpolypeptide (e.g., of a SARS-CoV-2-encoded S protein) that arecharacterized, when administered to a relevant population of adults, todisplay certain characteristics (e.g., achieve certain effects) asdescribed herein. In some embodiments, provided compositions may havebeen prepared, stored, transported, characterized, and/or used underconditions where temperature does not exceed a particular threshold.Alternatively or additionally, in some embodiments, providedcompositions may have been protected from light (e.g., from certainwavelengths) during some or all of their preparation, storage,transport, characterization, and/or use. In some embodiments, one ormore features of provided compositions (e.g., mRNA stability, as may beassessed, for example, by one or more of size, presence of particularmoiety or modification, etc; lipid nanoparticle stability oraggregation, pH, etc) may be or have been assessed at one or more pointsduring preparation, storage, transport, and/or use prior toadministration.

Among other things, the present disclosure documents that certainprovided compositions in which nucleotides within an mRNA are notmodified (e.g., are naturally-occurring A, U, C, G), and/or providedmethods relating to such compositions, are characterized (e.g., whenadministered to a relevant population, which may in some embodiments beor comprise an adult population), by an intrinsic adjuvant effect. Insome embodiments, such composition and/or method can induce an antibodyand/or a T cell response. In some embodiments, such a composition and/ormethod can induce a higher T cell response, as compared to conventionalvaccines (e.g., non-mRNA vaccines such as protein vaccines).

Alternatively or additionally, the present disclosure documents thatprovided compositions (e.g., compositions comprising a lipidnanoparticle encapsulated mRNA encoding at least a portion (e.g., thatis or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g.,of a SARS-CoV-2-encoded S protein)) in which nucleotides within an mRNAare modified, and/or provided methods relating to such compositions, arecharacterized (e.g., when administered to a relevant population, whichmay in some embodiments be or comprise an adult population), by absenceof an intrinsic adjuvant effect, or by a reduced intrinsic adjuvanteffect as compared with an otherwise comparable composition (or method)with unmodified results.

Alternatively or additionally, in some embodiments, such compositions(or methods) are characterized in that they (e.g., when administered toa relevant population, which may in some embodiments be or comprise anadult population) induce an antibody response and/or a CD4+ T cellresponse. Still further alternatively or additionally, in someembodiments, such compositions (or methods) are characterized in thatthey (e.g., when administered to a relevant population, which may insome embodiments be or comprise an adult population) induce a higherCD4+ T cell response than that observed with an alternative vaccineformat (e.g., a peptide vaccine). In some embodiments involving modifiednucleotides, such modified nucleotides may be present, for example, in a3′ UTR sequence, an antigen-encoding sequence, and/or a 5′UTR sequence.In some embodiments, modified nucleotides are or include one or moremodified uracil residues and/or one or more modified cytosine residues.

Among other things, the present disclosure documents that provided(e.g., compositions comprising a lipid nanoparticle encapsulated mRNAencoding at least a portion (e.g., that is or comprises an epitope) of aSARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded Sprotein)) and/or methods are characterized by (e.g., when administeredto a relevant population, which may in some embodiments be or comprisean adult population) sustained expression of an encoded polypeptide(e.g., of a SARS-CoV-2-encoded protein [such as an S protein] or portionthereof, which portion, in some embodiments, may be or comprise anepitope thereof). For example, in some embodiments, such compositionsand/or methods are characterized in that, when administered to a human,they achieve detectable polypeptide expression in a biological sample(e.g., serum) from such human and, in some embodiments, such expressionpersists for a period of time that is at least at least 36 hours orlonger, including, e.g., at least 48 hours, at least 60 hours, at least72 hours, at least 96 hours, at least 120 hours, at least 148 hours, orlonger.

Those skilled in the art, reading the present disclosure, willappreciate that it describes various mRNA constructs encoding at least aportion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encodedpolypeptide (e.g., of a SARS-CoV-2-encoded S protein)). Such person ofordinary skill, reading the present disclosure, will particularlyappreciate that it describes various mRNA constructs encoding at least aportion of a SARS-CoV-2 S protein, for example at least an RBD portionof a SARS-CoV-2 S protein. Still further, such a person of ordinaryskill, reading the present disclosure, will appreciate that it describesparticular characteristics and/or advantages of mRNA constructs encodingat least a portion (e.g., that is or comprises an epitope) of aSARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded Sprotein). Among other things, the present disclosure particularlydocuments surprising and useful characteristics and/or advantages ofcertain mRNA constructs encoding a SARS-CoV-2 RBD portion and, in someembodiments, not encoding a full length SARS-CoV-2 S protein. Withoutwishing to be bound by any particular theory, the present disclosuresuggests that provided mRNA constructs that encode less than afull-length SARS-CoV-2 S protein, and particularly those that encode atleast an RBD portion of such SARS-CoV-2 S protein may be particularlyuseful and/or effective for use as or in an immunogenic composition(e.g., a vaccine), and/or for achieving immunological effects asdescribed herein (e.g., generation of SARS-CoV-2 neutralizingantibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cellresponses)). In some embodiments, the present disclosure provides an RNA(e.g., mRNA) comprising an open reading frame encoding a polypeptidethat comprises a receptor-binding portion of a SARS-CoV-2 S protein,which RNA is suitable for intracellular expression of the polypeptide.In some embodiments, such an encoded polypeptide does not comprise thecomplete S protein. In some embodiments, the encoded polypeptidecomprises the receptor binding domain (RBD), for example, as shown inSEQ ID NO: 5. In some embodiments, the encoded polypeptide comprises thepeptide according to SEQ ID NO: 29 or 31. In some embodiments, such anRNA (e.g., mRNA) may be complexed by a (poly)cationic polymer,polyplex(es), protein(s) or peptide(s). In some embodiments, such an RNAmay be formulated in a lipid nanoparticle (e.g., ones described herein).In some embodiments, such an RNA (e.g., mRNA) may be particularly usefuland/or effective for use as or in an immunogenic composition (e.g., avaccine), and/or for achieving immunological effects as described herein(e.g., generation of SARS-CoV-2 neutralizing antibodies, and/or T cellresponses (e.g., CD4+ and/or CD8+ T cell responses)). In someembodiments, such an RNA (e.g., mRNA) may be useful for vaccinatinghumans (including, e.g., humans known to have been exposed and/orinfected by SARS-CoV-2, and/or humans not known to have been exposed toSARS-CoV-2).

Those skilled in the art, reading the present disclosure, will furtherappreciate that it describes various mRNA constructs comprising anucleic acid sequence that encodes a full-length SARS-CoV-2 Spikeprotein (e.g., including embodiments in which such encoded SARS-CoV-2Spike protein may comprise at least one or more amino acidsubstitutions, e.g., proline substitutions as described herein, and/orembodiments in which the mRNA sequence is codon-optimized e.g., formammalian, e.g., human, subjects). In some embodiments, such afull-length SARS-CoV-2 Spike protein may have an amino acid sequencethat is or comprises that set forth in SEQ ID NO: 7. Still further, sucha person of ordinary skill, reading the present disclosure, willappreciate, among other things, that it describes particularcharacteristics and/or advantages of certain mRNA constructs comprisinga nucleic acid sequence that encodes a full-length SARS-CoV-2 Spikeprotein. Without wishing to be bound by any particular theory, thepresent disclosure suggests that provided mRNA constructs that encode afull-length SARS-CoV-2 S protein may be particularly useful and/oreffective for use as or in an immunogenic composition (e.g., a vaccine)in particular subject population (e.g., particular age populations). Forexample, in some embodiments, such an mRNA composition may beparticularly useful in younger (e.g., less than 25 years old, 20 yearsold, 18 years old, 15 years, 10 years old, or lower) subjects;alternatively or additionally, in some embodiments, such an mRNAcomposition may be particularly useful in elderly subjects (e.g., over55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80years old, 85 years old, or higher). In particular embodiments, animmunogenic composition comprising such an mRNA construct providedherein exhibits a minimal to modest increase (e.g., no more than 30%increase, no more than 20% increase, or no more than 10% increase, orlower) in dose level and/or dose number-dependent systemicreactogenicity (e.g., fever, fatigue, headache, chills, diarrhea, musclepain, and/or joint pain, etc.) and/or local tolerability (e.g., pain,redness, and/or swelling, etc.), at least in some subjects (e.g., insome subject age groups); in some embodiments, such reactogenicityand/or local tolerability is observed particularly, in in younger agegroup (e.g., less than 25 years old, 20 years old, 18 years old orlower) subjects, and/or in older (e.g., elderly) age group (e.g., 65-85years old). In some embodiments, provided mRNA constructs that encode afull-length SARS-CoV-2 S protein may be particularly useful and/oreffective for use as or in an immunogenic composition (e.g., a vaccine)for inducing SARS-CoV-2 neutralizing antibody response level in apopulation of subjects that are at high risk for severe diseasesassociated with SARS-CoV-2 infection (e.g., an elderly population, forexample, 65-85 year-old group). In some embodiments, a person ofordinary skill, reading the present disclosure, will appreciate, amongother things, that provided mRNA constructs that encode a full-lengthSARS-CoV-2 S protein, which exhibit a favorable reactogenicity profile(e.g., as described herein) in younger and elderly age populations, maybe particularly useful and/or effective for use as or in an immunogeniccomposition (e.g., a vaccine) for achieving immunological effects asdescribed herein (e.g., generation of SARS-CoV-2 neutralizingantibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cellresponses)). In some embodiments, the present disclosure also suggeststhat provided mRNA constructs that encode a full-length SARS-CoV-2 Sprotein may be particularly effective to protect against SARS-CoV-2infection, as characterized by earlier clearance of SARS-CoV-2 viral RNAin non-human mammalian subjects (e.g., Rhesus macaques) that wereimmunized with immunogenic compositions comprising such mRNA constructsand subsequently challenged by SARS-CoV-2 strain. In some embodiments,such earlier clearance of SARS-CoV-2 viral RNA may be observed in thenose of non-human mammalian subjects (e.g., Rhesus macaques) that wereimmunized with immunogenic compositions comprising such mRNA constructsand subsequently challenged by SARS-CoV-2 strain.

In some embodiments, the present disclosure provides an RNA (e.g., mRNA)comprising an open reading frame encoding a full-length SARS-CoV-2 Sprotein (e.g., a full-length SARS-CoV-2 S protein with one or more aminoacid substitutions), which RNA is suitable for intracellular expressionof the polypeptide. In some embodiments, the encoded polypeptidecomprises the amino acid sequence of SEQ ID NO: 7. In some embodiments,such an RNA (e.g., mRNA) may be complexed by a (poly)cationic polymer,polyplex(es), protein(s) or peptide(s). In some embodiments, such an RNAmay be formulated in a lipid nanoparticle (e.g., ones described herein).

In some embodiments, an immunogenic composition provided herein maycomprise a plurality of (e.g., at least two or more, including, e.g., atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, etc.) immunoreactiveepitopes of a SARS-CoV-2 polypeptide or variants thereof. In some suchembodiments, such a plurality of immunoreactive epitopes may be encodedby a plurality of RNAs (e.g., mRNAs). In some such embodiments, such aplurality of immunoreactive epitopes may be encoded by a single RNA(e.g., mRNA). In some embodiments, nucleic acid sequences encoding aplurality of immunoreactive epitopes may be separated from each other ina single RNA (e.g., mRNA) by a linker (e.g., a peptide linker in someembodiments). Without wishing to be bound by any particular theory, insome embodiments, provided polyepitope immunogenic compositions(including, e.g., those that encode a full-length SARS-CoV-2 spikeprotein) may be particularly useful, when considering the geneticdiversity of SARS-CoV-2 variants, to provide protection against numerousviral variants and/or may offer a greater opportunity for development ofa diverse and/or otherwise robust (e.g., persistent, e.g., detectableabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days afteradministration of one or more doses) neutralizing antibody and/or T cellresponse, and in particular a particularly robust T_(H)1-type T cell(e.g., CD4+ and/or CD8+ T cell) response.

In some embodiments, the present disclosure documents that providedcompositions and/or methods are characterized by (e.g., whenadministered to a relevant population, which may in some embodiments beor comprise an adult population) in that they achieve one or moreparticular therapeutic outcomes (e.g., effective immune responses asdescribed herein and/or detectable expression of encoded SARS-CoV-2 Sprotein or an immunogenic fragment thereof) with a singleadministration; in some such embodiments, an outcome may be assessed,for example, as compared to that observed in absence of mRNA vaccinesdescribed herein. In some embodiments, a particular outcome may beachieved at a lower dose than required for one or more alternativestrategies.

In some embodiments, the present disclosure provides an immunogeniccomposition comprising an isolated messenger ribonucleic acid (mRNA)polynucleotide, wherein the isolated mRNA polynucleotide comprises anopen reading frame encoding a polypeptide that comprises areceptor-binding portion of a SARs-CoV-2 S protein, and wherein theisolated mRNA polynucleotide is formulated in at least one lipidnanoparticle. For example, in some embodiments, such a lipidnanoparticle may comprise a molar ratio of 20-60% ionizable cationiclipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol orsteroid, and 0.5-15% polymer-conjugated lipid (e.g., PEG-modifiedlipid). In some embodiments, a sterol or steroid included in a lipidnanoparticle may be or comprise cholesterol. In some embodiments, aneutral lipid may be or comprise1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments,a polymer-conjugated lipid may be or comprise PEG2000 DMG. In someembodiments, such an immunogenic composition may comprise a total lipidcontent of about 1 mg to 10 mg, or 3 mg to 8 mg, or 4 mg to 6 mg. Insome embodiments, such an immunogenic composition may comprise a totallipid content of about 5 mg/mL-15 mg/mL or 7.5 mg/mL-12.5 mg/mL or 9-11mg/mL. In some embodiments, such an isolated mRNA polynucleotide isprovided in an effective amount to induce an immune response in asubject administered at least one dose of the immunogenic composition.In some embodiments, a polypeptide encoded by a provided isolated mRNApolynucleotide does not comprise the complete S protein. In someembodiments, such an isolated mRNA polynucleotide provided in animmunogenic composition is not self-replicating RNA.

In some embodiments, an immune response may comprise generation of abinding antibody titer against SARS-CoV-2 protein (including, e.g., astabilized prefusion spike trimer in some embodiments) or a fragmentthereof. In some embodiments, an immune response may comprise generationof a binding antibody titer against the receptor binding domain (RBD) ofthe SARS-CoV-2 spike protein. In some embodiments, a providedimmunogenic composition has been established to achieve a detectablebinding antibody titer after administration of a first dose, withseroconversion in at least 70% (including, e.g., at least 80%, at least90%, at least 95% and up to 100%) of a population of subjects receivingsuch a provided immunogenic composition, for example, by about 2 weeks.

In some embodiments, an immune response may comprise generation of aneutralizing antibody titer against SARS-CoV-2 protein (including, e.g.,a stabilized prefusion spike trimer in some embodiments) or a fragmentthereof. In some embodiments, an immune response may comprise generationof a neutralizing antibody titer against the receptor binding domain(RBD) of the SARS-CoV-2 spike protein. In some embodiments, a providedimmunogenic composition has been established to achieve a neutralizingantibody titer in an appropriate system (e.g., in a human infected withSARS-CoV-2 and/or a population thereof, and/or in a model systemtherefor). For example, in some embodiments, such neutralizing antibodytiter may have been demonstrated in one or more of a population ofhumans, a non-human primate model (e.g., Rhesus macaques), and/or amouse model.

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce viralinfection of B cells relative to that observed for an appropriatecontrol (e.g., an unvaccinated control subject, or a subject vaccinatedwith a live attenuated viral vaccine, an inactivated viral vaccine, or aprotein subunit viral vaccine, or a combination thereof). In some suchembodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce the rate ofasymptomatic viral infection relative to that observed for anappropriate control (e.g., an unvaccinated control subject, or a subjectvaccinated with a live attenuated viral vaccine, an inactivated viralvaccine, or a protein subunit viral vaccine, or a combination thereof).In some such embodiments, such reduction is of at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In someembodiments, such reduction can be characterized by assessment ofSARS-CoV-2 N protein serology. Significant protection againstasymptomatic infection was also confirmed by real life observations (seealso: Dagan N. et al., N Engl J Med. 2021, doi: 10.1056/NEJMoa2101765.Epub ahead of print. PMID: 33626250)

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce or blockfusion of virus with epithelial cells and/or B cells of a vaccinatedsubject relative to that observed for an appropriate control (e.g., anunvaccinated control subject, or a subject vaccinated with a liveattenuated viral vaccine, an inactivated viral vaccine, or a proteinsubunit viral vaccine, or a combination thereof). In some suchembodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

In some embodiments, induction of a neutralizing antibody titer may becharacterized by an elevation in the number of B cells, which in someembodiments may include plasma cells, class-switched IgG1- andIgG2-positive B cells, and/or germinal center B cells. In someembodiments, a provided immunogenic composition has been established toachieve such an elevation in the number of B cells in an appropriatesystem (e.g., in a human infected with SARS-CoV-2 and/or a populationthereof, and/or in a model system therefor). For example, in someembodiments, such an elevation in the number of B cells may have beendemonstrated in one or more of a population of humans, a non-humanprimate model (e.g., Rhesus macaques), and/or a mouse model. In someembodiments, such an elevation in the number of B cells may have beendemonstrated in draining lymph nodes and/or spleen of a mouse modelafter (e.g., at least 7 days, at least 8 days, at least 9 days, at least10 days, at least 11 days, at least 12 days, at least 13 days, at least14 days, after) immunization of such a mouse model with a providedimmunogenic composition.

In some embodiments, induction of a neutralizing antibody titer may becharacterized by a reduction in the number of circulating B cells inblood. In some embodiments, a provided immunogenic composition has beenestablished to achieve such a reduction in the number of circulating Bcells in blood of an appropriate system (e.g., in a human infected withSARS-CoV-2 and/or a population thereof, and/or in a model systemtherefor). For example, in some embodiments, such a reduction in thenumber of circulating B cells in blood may have been demonstrated in oneor more of a population of humans, a non-human primate model (e.g.,Rhesus macaques), and/or a mouse model. In some embodiments, such areduction in the number of circulating B cells in blood may have beendemonstrated in a mouse model after (e.g., at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 8 days, at least 9days, at least 10 days, after) immunization of such a mouse model with aprovided immunogenic composition. Without wishing to be bound by theory,a reduction in circulating B cells in blood may be due to B cell homingto lymphoid compartments.

In some embodiments, an immune response induced by a providedimmunogenic composition may comprise an elevation in the number of Tcells. In some embodiments, such an elevation in the number of T cellsmay include an elevation in the number of T follicular helper (T_(FH))cells, which in some embodiments may comprise one or more subsets withICOS upregulation. One of skilled in the art will understand thatproliferation of T_(FH) in germinal centres is integral for generationof an adaptive B-cell response, and also that in humans, T_(FH)occurring in the circulation after vaccination is typically correlatedwith a high frequency of antigen-specific antibodies. In someembodiments, a provided immunogenic composition has been established toachieve such an elevation in the number of T cells (e.g., T_(FH) cells)in an appropriate system (e.g., in a human infected with SARS-CoV-2and/or a population thereof, and/or in a model system therefor). Forexample, in some embodiments, such an elevation in the number of T cells(e.g., T_(FH) cells) may have been demonstrated in one or more of apopulation of humans, a non-human primate model (e.g., Rhesus macaques),and/or a mouse model. In some embodiments, such an elevation in thenumber of T cells (e.g., e.g., T_(FH) cells) may have been demonstratedin draining lymph nodes, spleen, and/or blood of a mouse model after(e.g., at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 14 days, after)immunization of such a mouse model with a provided immunogeniccomposition. In some embodiments, a protective response againstSARS-CoV-2 induced by a provided immunogenic composition has beenestablished in an appropriate model system for SARS-CoV-2. For example,in some embodiments, such a protective response may have beendemonstrated in an animal model, e.g., a non-human primate model (e.g.,Rhesus macaques) and/or a mouse model. In some embodiments, a non-humanprimate (e.g., Rhesus macaque) or a polulation thereof that has/havereceived at least one immunization with a provided immunogeniccomposition is/are challenged with SARS-CoV-2, e.g., through intranasaland/or intratracheal route. In some embodiments, such a challenge may beperformed several weeks (e.g., 5-10 weeks) after at least oneimmunization (including, e.g., at least two immunizations) with aprovided immunogenic composition. In some embodiments, such a challengemay be performed when a detectable level of a SARS-CoV-2 neutralizingtiter (e.g., antibody response to SARS-CoV-2 spike protein and/or afragment thereof, including, e.g., but not limited to a stabilizedprefusion spike trimer, S-2P, and/or antibody response toreceptor-binding portion of SARS-CoV-2) is achieved in non-humanprimate(s) (e.g., Rhesus macaque(s)) that has received at least oneimmunization (including, e.g., at least two immunizations) with aprovided immunogenic composition. In some embodiments, a protectiveresponse is characterized by absence of or reduction in detectable viralRNA in bronchoalveolar lavage (BAL) and/or nasal swabs of challengednon-human primate(s) (e.g., Rhesus macaque(s)). In some embodiments,immunogenic compositions described herein may have been characterized inthat a larger percent of challenged animals, for example, non-humanprimates in a population (e.g., Rhesus macaques), that have received atleast one immunization (including, e.g., at least two immunizations)with a provided immunogenic composition display absence of detectableRNA in their BAL and/or nasal swab, as compared to a population ofnon-immunized animals, for example, non-human primates (e.g., Rhesusmacaques). In some embodiments, immunogenic compositions describedherein may have been characterized in that challenged animals, forexample, non-human in a population (e.g., Rhesus macaques), that havereceived at least one immunization (including, e.g., at least twoimmunizations) with a provided immunogenic composition may showclearance of viral RNA in nasal swab no later than 10 days, including,e.g., no later than 8 days, no later than 6 days, no later than 4 days,etc., as compared to a population of non-immunized animals, for example,non-human primates (e.g., Rhesus macaques).

In some embodiments, immunogenic compositions described herein whenadministered to subjects in need thereof do not substantially increasethe risk of vaccine-associated enhanced respiratory disease. In someembodiments, such vaccine-associated enhanced respiratory disease may beassociated with antibody-dependent enhancement of replication and/orwith vaccine antigens that induced antibodies with poor neutralizingactivity and Th2-biased responses. In some embodiments, immunogeniccompositions described herein when administered to subjects in needthereof do not substantially increase the risk of antibody-dependentenhancement of replication.

In some embodiments, a single dose of an mRNA composition (e.g.,formulated in lipid nanoparticles) can induce a therapeutic antibodyresponse in less than 10 days of vaccination. In some embodiments, sucha therapeutic antibody response may be characterized in that when suchan mRNA vaccine can induce production of about 10-100 ug/mL IgG measuredat 10 days after vaccination at a dose of 0.1 to 10 ug or 0.2-5 ug in ananimal model. In some embodiments, such a therapeutic antibody responsemay be characterized in that such an mRNA vaccine induces about 100-1000ug/mL IgG measured at 20 days of vaccination at a dose of 0.1 to 10 ugor 0.2-5 ug in an animal model. In some embodiments, a single dose mayinduce a pseudovirus-neutralization titer, as measured in an animalmodel, of 10-200 pVN50 titer 15 days after vaccination. In someembodiments, a single dose may induce a pseudovirus-neutralizationtiter, as measured in an animal model, of 50-500 pVN50 titer 15 daysafter vaccination.

In some embodiments, a single dose of an mRNA composition can expandantigen-specific CD8 and/or CD4 T cell response by at least at 50% ormore (including, e.g., at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or more), as compared to that observed inabsence of such an mRNA construct encoding a SARS-COV2 immunogenicprotein or fragment thereof (e.g., spike protein and/or receptor bindingdomain). In some embodiments, a single dose of an mRNA composition canexpand antigen-specific CD8 and/or CD4 T cell response by at least at1.5-fold or more (including, e.g., at least 2-fold, at least 3-fold, atleast 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 500-fold, at least 1000-fold, or more), as compared to thatobserved in absence of such an mRNA construct encoding a SARS-COV2immunogenic protein or fragment thereof (e.g., spike protein and/orreceptor binding domain).

In some embodiments, a regimen (e.g., a single dose of an mRNAcomposition) can expand T cells that exhibit a Th1 phenotype (e.g., ascharacterized by expression of IFN-gamma, IL-2, IL-4, and/or IL-5) by atleast at 50% or more (including, e.g., at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or more), as compared to thatobserved in absence of such an mRNA construct encoding a SARS-COV2immunogenic protein or fragment thereof (e.g., spike protein and/orreceptor binding domain). In some embodiments, a regimen (e.g., a singledose of an mRNA composition) can expand T cells that exhibit a Th1phenotype (e.g., as characterized by expression of IFN-gamma, IL-2,IL-4, and/or IL-5), for example by at least at 1.5-fold or more(including, e.g., at least 2-fold, at least 3-fold, at least 5-fold, atleast 10-fold, at least 50-fold, at least 100-fold, at least 500-fold,at least 1000-fold, or more), as compared to that observed in absence ofsuch an mRNA construct encoding a SARS-COV2 immunogenic protein orfragment thereof (e.g., spike protein and/or receptor binding domain).In some embodiments, a T-cell phenotype may be or comprise aTh1-dominant cytokine profile (e.g., as characterized by INF-gammapositive and/or IL-2 positive), and/or no by or biologicallyinsignificant IL-4 secretion.

In some embodiments, a regimen as described herein (e.g., one or moredoses of an mRNA composition) induces and/or achieves production ofRBD-specific CD4+ T cells. Among other things, the present disclosuredocuments that mRNA compositions encoding an RBD-containing portion of aSARS-CoV-2 spike protein (e.g., and not encoding a full-lengthSARS-CoV-2 spike protein) may be particularly useful and/or effective insuch induction and/or production of RBD-specific CD4+ T cells. In someembodiments, RBD-specific CD4+ T-cells induced by an mRNA compositiondescribed herein (e.g., by an mRNA composition that encodings anRBD-containing-portion of a SARS-CoV-2 spike protein and, in someembodiments not encoding a full-length SARS-CoV-2 spike protein)demonstrate a Th1-dominant cytokine profile (e.g., as characterized byINF-gamma positive and/or IL-2 positive), and/or by no or biologicallyinsignificant IL-4 secretion.

In some embodiments, characterization of CD4+ and/or CD8+ T cellresponses (e.g., described herein) in subjects receiving mRNAcompositions (e.g., as described herein) may be performed using ex vivoassays using PBMCs collected from the subjects, e.g., assays asdescribed in the Examples.

In some embodiments, immunogenicity of mRNA compositions describedherein may be assessed by one of or more of the following serologicalimmunogenicity assays: detection of IgG, IgM, and/or IgA to SARS-CoV-2 Sprotein present in blood samples of a subject receiving a provided mRNAcomposition, and/or neutralization assays using SARS-CoV-2 pseudovirusand/or a wild-type SARS-CoV-2 virus.

In some embodiments, an mRNA composition (e.g., as described herein)provide a relatively low adverse effect (e.g., Grade 1-Grade 2 pain,redness and/or swelling) within 7 days after vaccinations at a dose of10 ug-100 ug or 1 ug-50 ug. In some embodiments, mRNA compositions(e.g., as described herein) provide a relatively low observation ofsystemic events (e.g., Grade 1-Grade 2 fever, fatigue, headache, chills,vomiting, diarrhea, muscle pain, joint pain, medication, andcombinations thereof) within 7 days after vaccinations at a dose of 10ug-100 ug.

In some embodiments, mRNA compositions are characterized in that whenadministered to subjects at 10-100 ug dose or 1 ug-50 ug, IgG directedto a SARS-CoV2 immunogenic protein or fragment thereof (e.g., spikeprotein and/or receptor binding domain) may be produced at a level of100-100,000 U/mL or 500-50,000 U/mL 21 days after vaccination.

In some embodiments, an mRNA encodes a natively-folded trimeric receptorbinding protein of SARS-CoV-2. In some embodiments, an mRNA encodes avariant of such receptor binding protein such that the encoded variantbinds to ACE2 at a Kd of 10 pM or lower, including, e.g., at a Kd of 9pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, or lower. In some embodiments, an mRNAencodes a variant of such receptor binding protein such that the encodedvariant binds to ACE2 at a Kd of 5 pM. In some embodiments, an mRNAencodes a trimeric receptor binding portion of SARS-CoV-2 that comprisesan ACE2 receptor binding site. In some embodiments, an mRNA comprises acoding sequence for a receptor-binding portion of SARS-CoV-2 and atrimerization domain (e.g., a natural trimerization domain (foldon) ofT4 fibritin) such that the coding sequence directs expression of atrimeric protein that has an ACE2 receptor binding site and binds ACE2.In some embodiments, an mRNA encodes a trimeric receptor binding portionof SARS-CoV-2 or a variant thereof such that its Kd is smaller than thatfor a monomeric receptor-binding domain (RBD) of SARS-CoV-2. Forexample, in some embodiments, an mRNA encodes a trimeric receptorbinding portion of SARS-CoV-2 or a variant thereof such that its Kd isat least 10-fold (including, e.g., at least 50-fold, at least 100-fold,at least 500-fold, at least 1000-fold, etc.) smaller than that for a RBDof SARS-CoV-2.

In some embodiments, a trimer receptor binding portion of SARS-CoV-2encoded by an mRNA (e.g., as described herein) may be determined to havea size of about 3-4 angstroms when it is complexed with ACE2 and B⁰AT1neutral amino acid transporter in a closed conformation, ascharacterized by electron cryomicroscopy (cryoEM). In some embodiments,geometric mean SARS-CoV-2 neutralizing titer that characterizes and/oris achieved by an mRNA composition or method as described herein canreach at least 1.5-fold, including, at least 2-fold, at least 2.5-fold,at least 3-fold, or higher, that of a COVID-19 convalescent human panel(e.g., a panel of sera from COVID-19 convalescing humans obtained 20-40days after the onset of symptoms and at least 14 days after the start ofasymptomatic convalescence.

In some embodiments, mRNA compositions as provided herein may becharacterized in that subjects who have been treated with suchcompositions (e.g., with at least one dose, at least two doses, etc) mayshow reduced and/or more transient presence of viral RNA in relevantsite(s) (e.g., nose and/or lungs, etc, and/or any other tissuesusceptible to infection) as compared with an appropriate control (e.g.,an established expected level for a comparable subject or population nothaving been so treated and having been exposed to virus under reasonablycomparable exposure conditions)

In some embodiments, the RBD antigen expressed by an mRNA construct(e.g., as described herein) can be modified by addition of aT4-fibritin-derived “foldon” trimerization domain, for example, toincrease its immunogenicity.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that certain local reactions (e.g., pain, redness,and/or swelling, etc.) and/or systemic events (e.g., fever, fatigue,headache, etc.) may appear and/or peak at Day 2 after vaccination. Insome embodiments, mRNA compositions described herein are characterizedin that certain local reactions (e.g., pain, redness, and/or swelling,etc.) and/or systemic events (e.g., fever, fatigue, headache, etc.) mayresolve by Day 7 after vaccination.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that no Grade 1 or greater change in routineclinical laboratory values or laboratory abnormalities are observed insubjects receiving mRNA compositions (e.g., as described herein).Examples of such clinical laboratory assays may include lymphocytecount, hematological changes, etc.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that by 21 days after a first dose (e.g., 10-100 uginclusive or 1 ug-50 ug inclusive), geometric mean concentrations (GMCs)of IgG directed to a SARS-CoV-2 S polypeptide or an immunogenic fragmentthereof (e.g., RBD) may reach 200-3000 units/mL or 500-3000 units/mL or500-2000 units/mL, compared to 602 units/mL for a panel of COVID-19convalescent human sera. In some embodiments, mRNA compositionsdescribed herein are characterized in that by 7 days after a second dose(e.g., 10-30 ug inclusive; or 1 ug-50 ug inclusive), geometric meanconcentrations (GMCs) of IgG directed to a SARS-CoV-2 spike polypeptideor an immunogenic fragment thereof (e.g., RBD) may increase by at least8-fold or higher, including, e.g., at least 9-fold, at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, atleast 35-fold, at least 40-fold, or higher. In some embodiments, mRNAcompositions described herein are characterized in that by 7 days aftera second dose (e.g., 10-30 ug inclusive; or 1 ug-50 ug inclusive),geometric mean concentrations (GMCs) of IgG directed to a SARS-CoV-2 Spolypeptide or an immunogenic fragment thereof (e.g., RBD) may increaseto 1500 units/mL to 40,000 units/mL or 4000 units/mL to 40,000 units/mL.In some embodiments, antibody concentrations described herein canpersist to at least 20 days or longer, including, e.g., at least 25days, at least 30 days, at least 35 days, at least 40 days, at least 45days, at least 50 days, after a first dose, or at least 10 days orlonger, including, e.g., at least 15 days, at least 20 days, at least 25days, or longer, after a second dose. In some embodiments, antibodyconcentrations can persist to 35 days after a first dose, or at least 14days after a second dose.

In some embodiments, mRNA compositions described herein arecharacterized in that when measured at 7 days after a second dose (e.g.,1-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptide oran immunogenic fragment thereof (e.g., RBD) is at least 30% higher(including, e.g., at least 40% higher, at least 50% higher, at least60%, higher, at least 70% higher, at least 80% higher, at least 90%higher, at least 95% higher, as compared to antibody concentrationsobserved in a panel of COVID-19 convalescent human serum. In manyembodiments, geometric mean concentration (GMC) of IgG described hereinis GMCs of RBD-binding IgG.

In some embodiments, mRNA compositions described herein arecharacterized in that when measured at 7 days after a second dose (e.g.,10-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptideor an immunogenic fragment thereof (e.g., RBD) is at least 1.1-foldhigher (including, e.g., at least 1.5-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold higher, atleast 7-fold higher, at least 8-fold higher, at least 9-fold higher, atleast 10-fold higher, at least 15-fold higher, at least 20-fold higher,at least 25-fold higher, at least 30-fold higher), as compared toantibody concentrations observed in a panel of COVID-19 convalescenthuman serum, In many embodiments, geometric mean concentration (GMC) ofIgG described herein is GMCs of RBD-binding IgG.

In some embodiments, mRNA compositions described herein arecharacterized in that when measured at 21 days after a second dose, GMCof IgG directed to a SARS-CoV-2 S polypeptide or an immunogenic fragmentthereof (e.g., RBD) is at least 5-fold higher (including, e.g., at least6-fold higher, at least 7-fold higher, at least 8-fold higher, at least9-fold higher, at least 10-fold higher, at least 15-fold higher, atleast 20-fold higher, at least 25-fold higher, at least 30-fold higher),as compared to antibody concentrations observed in a panel of COVID-19convalescent human serum, In many embodiments, geometric meanconcentration (GMC) of IgG described herein is GMCs of RBD-binding IgG.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that an increase (e.g., at least 30%, at least 40%,at least 50%, or more) in SARS-CoV-2 neutralizing geometric mean titers(GMTs) is observed 21 days after a first dose. In some embodiments, mRNAcompositions described herein are characterized in that a substantiallygreater serum neutralizing GMTs are achieved 7 days after subjectsreceive a second dose (e.g., 10 μg-30 μg inclusive), reaching 150-300,compared to 94 for a COVID-19 convalescent serum panel.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that 7 days after administration of the seconddose, the protective efficacy is at least 60%, e.g., at least 70%, atleast 80%, at least 90, or at least 95%. In one embodiment, mRNAcompositions and/or methods described herein are characterized in that 7days after administration of the second dose, the protective efficacy isat least 70%. In one embodiment, mRNA compositions and/or methodsdescribed herein are characterized in that 7 days after administrationof the second dose, the protective efficacy is at least 80%. In oneembodiment, mRNA compositions and/or methods described herein arecharacterized in that 7 days after administration of the second dose,the protective efficacy is at least 90%. In one embodiment, mRNAcompositions and/or methods described herein are characterized in that 7days after administration of the second dose, the protective efficacy isat least 95%.

In some embodiments, an RNA composition provided herein is characterizedin that it induces an immune response against SARS-CoV-2 after at least7 days after a dose (e.g., after a second dose). In some embodiments, anRNA composition provided herein is characterized in that it induces animmune response against SARS-CoV-2 in less than 14 days after a dose(e.g., after a second dose). In some embodiments, an RNA compositionprovided herein is characterized in that it induces an immune responseagainst SARS-CoV-2 after at least 7 days after a vaccination regimen. Insome embodiments, a vaccination regimen comprises a first dose and asecond dose. In some embodiments, a first dose and a second dose areadministered by at least 21 days apart. In some such embodiments, animmune response against SARS-CoV-2 is induced at least after 28 daysafter a first dose.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that geometric mean concentration (GMCs) ofantibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenicfragment thereof (e.g., RBD), as measured in serum from subjectsreceiving mRNA compositions of the present disclosure (e.g., at a doseof 10-30 ug inclusive), is substantially higher than in a convalescentserum panel (e.g., as described herein). In some embodiments where asubject may receive a second dose (e.g., 21 days after 1 first dose),geometric mean concentration (GMCs) of antibodies directed to aSARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g.,RBD), as measured in serum from the subject, may be 8.0-fold to 50-foldhigher than a convalescent serum panel GMC. In some embodiments where asubject may receive a second dose (e.g., 21 days after 1 first dose),geometric mean concentration (GMCs) of antibodies directed to aSARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g.,RBD), as measured in serum from the subject, may be at least 8.0-fold orhigher, including, e.g., at least 10-fold, at least 20-fold, at least30-fold, at least 40-fold, at least 50-fold, at least 60-fold or higher,as compared to a convalescent serum panel GMC.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that the SARS-CoV-2 neutralizing geometric meantiter, as measured at 28 days after a first dose or 7 days after asecond dose, may be at least 1.5-fold or higher (including, e.g., atleast 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold orhigher), as compared to a neutralizing GMT of a convalescent serumpanel.

In some embodiments, a regimen administered to a subject may be orcomprise a single dose. In some embodiments, a regimen administered to asubject may comprise a plurality of doses (e.g., at least two doses, atleast three doses, or more). In some embodiments, a regimen administeredto a subject may comprise a first dose and a second dose, which aregiven at least 2 weeks apart, at least 3 weeks apart, at least 4 weeksapart, or more. In some embodiments, such doses may be at least 1 month,at least 2 months, at least 3 months, at least 4 months, at least 5months, at least 6 months, at least 7 months, at least 8 months, atleast 9 months, at least 10 months, at least 11 months, at least 12months, or more apart. In some embodiments, doses may be administereddays apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In someembodiments, doses may be administered about 1 to about 3 weeks apart,or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, orabout 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart.In some embodiments, doses may be separated by a period of about 7 toabout 60 days, such as for example about 14 to about 48 days, etc. Insome embodiments, a minimum number of days between doses may be about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21or more. In some embodiments, a maximum number of days between doses maybe about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, or fewer. In some embodiments, doses may beabout 21 to about 28 days apart. In some embodiments, doses may be about19 to about 42 days apart. In some embodiments, doses may be about 7 toabout 28 days apart. In some embodiments, doses may be about 14 to about24 days. In some embodiments, doses may be about 21 to about 42 days.

In some embodiments, particularly for compositions established toachieve elevated antibody and/or T-cell titres for a period of timelonger than about 3 weeks—e.g., in some embodiments, a providedcomposition is established to achieve elevated antibody and/or T-celltitres (e.g., specific for a relevant portion of a SARS-CoV-2 spikeprotein) for a period of time longer than about 3 weeks; in some suchembodiments, a dosing regimen may involve only a single dose, or mayinvolve two or more doses, which may, in some embodiments, be separatedfrom one another by a period of time that is longer than about 21 daysor three weeks. For example, in some such embodiments, such period oftime may be about 4 weeks, 5 weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks,10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 wees, 16 weeks, 17weeks, 18 weeks, 19 weeks, 20 weeks or more, or about 1 month, 2 months,3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10, months, 11 months, 12 months or more, or in some embodiments about ayear or more. In some embodiments, a first dose and a second dose(and/or other subsequent dose) may be administered by intramuscularinjection. In some embodiments, a first dose and a second dose may beadministered in the deltoid muscle. In some embodiments, a first doseand a second dose may be administered in the same arm. In someembodiments, an mRNA composition described herein is administered (e.g.,by intramuscular injection) as a series of two doses (e.g., 0.3 mL each)21 days part. In some embodiments, each dose is about 30 ug. In someembodiments, each dose may be higher than 30 ug, e.g., about 40 ug,about 50 ug, about 60 ug. In some embodiments, each dose may be lowerthan 30 ug, e.g., about 20 ug, about 10 ug, about 5 ug, etc. In someembodiments, each dose is about 3 ug or lower, e.g., about 1 ug. In somesuch embodiments, an mRNA composition described herein is administeredto subjects of age 16 or older (including, e.g., 16-85 years). In somesuch embodiments, an mRNA composition described herein is administeredto subjects of age 18-55. In some such embodiments, an mRNA compositionescribed herein is administered to subjects of age 56-85. In someembodiments, an mRNA composition described herein is administered (e.g.,by intramuscular injection) as a single dose.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that RBD-specific IgG (e.g., polyclonal response)induced by such mRNA compositions and/or methods exhibit a higherbinding affinity to RBD, as compared to a reference human monoclonalantibody with SARS-CoV-2 RBD-binding affinity (e.g., CR3022 as describedin J. ter Meulen et al., PLOS Med. 3, e237 (2006).)

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity across a panel (e.g., at least 10, at least 15, ormore) of SARs-CoV-2 spike variants. In some embodiments, such SARs-CoV-2spike variants include mutations in RBD (e.g., but not limited to Q321L,V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K,K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., as compared toSEQ ID NO: 1), and/or mutations in spike protein (e.g., but not limitedto D614G, etc., as compared to SEQ ID NO: 1). Those skilled in the artare aware of various spike variants, and/or resources that document them(e.g., the Table of mutating sites in Spike maintained by the COVID-19Viral Genome Analysis Pipeline and found atcov.lanl.gov/components/sequence/COV/int_sites_tbls.comp) (last accessed24 Aug. 2020), and, reading the present specification, will appreciatethat mRNA compositions and/or methods described herein can becharacterized for there ability to induce sera in vaccinated subjectthat display neutralizing activity with respect to any or all of suchvariants and/or combinations thereof.

In particular embodiments, mRNA compositions encoding RBD of aSARS-CoV-2 spike protein are characterized in that sera of vaccinatedsubjects display neutralizing activity across a panel (e.g., at least10, at least 15, or more) of SARs-CoV-2 spike variants including RBDvariants (e.g., but not limited to Q321L, V341I, A348T, N354D, S359N,V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N,V483A, Y508H, H519P, etc., as compared to SEQ ID NO: 1) and spikeprotein variants (e.g., but not limited to D614G, as compared to SEQ IDNO: 1). In particular embodiments, mRNA compositions encoding aSARS-CoV-2 spike protein variant that includes two consecutive prolinesubstitutions at amino acid positions 986 and 987, at the top of thecentral helix in the S2 subunit, are characterized in that sera ofvaccinated subjects display neutralizing activity across a panel (e.g.,at least 10, at least 15, or more) of SARs-CoV-2 spike variantsincluding RBD variants (e.g., but not limited to Q321L, V341I, A348T,N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V,G476S, S477N, V483A, Y508H, H519P, etc., as compared to SEQ ID NO: 1)and spike protein variants (e.g., but not limited to D614G, as comparedto SEQ ID NO: 1). For example, in some embodiments, the mRNA compositionencoding SEQ ID NO: 7 (S P2) elicits an immune response against any oneof a SARs-CoV-2 spike variant including RBD variants (e.g., but notlimited to Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I,Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P,etc., as compared to SEQ ID NO: 1) and spike protein variants (e.g., butnot limited to D614G, as compared to SEQ ID NO: 1).

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at position 501 in spike protein as compared to SEQID NO: 1. In some embodiments, mRNA compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a N501Y mutation in spike protein as compared to SEQID NO: 1.

Said one or more SARs-CoV-2 spike variants including a mutation atposition 501 in spike protein as compared to SEQ ID NO: 1 or said one ormore SARs-CoV-2 spike variants including a N501Y mutation in spikeprotein as compared to SEQ ID NO: 1 may include one or more furthermutations as compared to SEQ ID NO: 1 (e.g., but not limited to H69/V70deletion, Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H,D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242/A243/L244 deletionetc., as compared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “Variant ofConcern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7). Thevariant had previously been named the first Variant Under Investigationin December 2020 (VUI-202012/01) by Public Health England, but wasreclassified to a Variant of Concern (VOC-202012/01). VOC-202012/01 is avariant of SARS-CoV-2 which was first detected in October 2020 duringthe COVID-19 pandemic in the United Kingdom from a sample taken theprevious month, and it quickly began to spread by mid-December. It iscorrelated with a significant increase in the rate of COVID-19 infectionin United Kingdom; this increase is thought to be at least partlybecause of change N501Y inside the spike glycoprotein's receptor-bindingdomain, which is needed for binding to ACE2 in human cells. TheVOC-202012/01 variant is defined by 23 mutations: 13 non-synonymousmutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17mutations that change proteins and six that do not). The spike proteinchanges in VOC 202012/01 include deletion 69-70, deletion 144, N501Y,A570D, D614G, P681H, T716I, S982A, and D1118H. One of the most importantchanges in VOC-202012/01 seems to be N501Y, a change from asparagine (N)to tyrosine (Y) at amino-acid site 501. This mutation alone or incombination with the deletion at positions 69/70 in the N terminaldomain (NTD) may enhance the transmissibility of the virus.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: deletion 69-70, deletion 144, N501Y, A570D, D614G,P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”. Thisvariant was first observed in samples from October 2020, and since thenmore than 300 cases with the 501.V2 variant have been confirmed by wholegenome sequencing (WGS) in South Africa, where in December 2020 it wasthe dominant form of the virus. Preliminary results indicate that thisvariant may have an increased transmissibility. The 501.V2 variant isdefined by multiple spike protein changes including: D80A, D215G, E484K,N501Y and A701V, and more recently collected viruses have additionalchanges: L18F, R246I, K417N, and deletion 242-244.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y and A701V as compared toSEQ ID NO: 1, and optionally: L18F, R246I, K417N, and deletion 242-244as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant may alsoinclude a D614G mutation as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a H69/V70 deletion in spike protein as compared to SEQ ID NO:1.

In some embodiments, one or more SARs-CoV-2 spike variants including aH69/V70 deletion in spike protein as compared to SEQ ID NO: 1 mayinclude one or more further mutations as compared to SEQ ID NO: 1 (e.g.,but not limited to Y144 deletion, N501Y, A570D, D614G, P681H, T716I,S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I etc., as comparedto SEQ ID NO: 1),

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “Variant ofConcern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: deletion 69-70, deletion 144, N501Y, A570D, D614G,P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “Cluster 5”, alsoreferred to as ΔFVI-spike by the Danish State Serum Institute (SSI). Itwas discovered in North Jutland, Denmark, and is believed to have beenspread from minks to humans via mink farms. In cluster 5, severaldifferent mutations in the spike protein of the virus have beenconfirmed. The specific mutations include 69-70deltaHV (a deletion ofthe histidine and valine residues at the 69th and 70th position in theprotein), Y453F (a change from tyrosine to phenylalanine at position453), I692V (isoleucine to valine at position 692), M1229I (methionineto isoleucine at position 1229), and optionally S1147L (serine toleucine at position 1147).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: deletion 69-70, Y453F, I692V, M1229I, andoptionally S1147L, as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at position 614 in spike protein as compared to SEQID NO: 1. In some embodiments, mRNA compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a D614G mutation in spike protein as compared to SEQID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 614 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a D614G mutation inspike protein as compared to SEQ ID NO: 1 may include one or morefurther mutations as compared to SEQ ID NO: 1 (e.g., but not limited toH69/V70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A,D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242/A243/L244deletion, Y453F, I692V, S1147L, M1229I etc., as compared to SEQ ID NO:1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “Variant ofConcern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: deletion 69-70, deletion 144, N501Y, A570D, D614G,P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at positions 501 and 614 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, mRNA compositions and/ormethods described herein are characterized in that sera of vaccinatedsubjects display neutralizing activity against one or more SARs-CoV-2spike variants including a N501Y mutation and a D614G mutation in spikeprotein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 501 and 614 in spike protein as compared to SEQ IDNO: 1 or said one or more SARs-CoV-2 spike variants including a N501Ymutation and a D614G mutation in spike protein as compared to SEQ ID NO:1 may include one or more further mutations as compared to SEQ ID NO: 1(e.g., but not limited to H69/V70 deletion, Y144 deletion, A570D, P681H,T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I etc., as comparedto SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “Variant ofConcern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: deletion 69-70, deletion 144, N501Y, A570D, D614G,P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at position 484 in spike protein as compared to SEQID NO: 1. In some embodiments, mRNA compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a E484K mutation in spike protein as compared to SEQID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 484 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a E484K mutation inspike protein as compared to SEQ ID NO: 1 may include one or morefurther mutations as compared to SEQ ID NO: 1 (e.g., but not limited toH69/V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H, T716I,S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242/A243/L244deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T,H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, and A701V, as comparedto SEQ ID NO: 1, and optionally: L18F, R246I, K417N, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

Lineage B.1.1.248, known as the Brazil(ian) variant, is one of thevariants of SARS-CoV-2 which has been named P.1 lineage and has 17unique amino acid changes, 10 of which in its spike protein, includingN501Y and E484K. B.1.1.248 originated from B.1.1.28. E484K is present inboth B.1.1.28 and B.1.1.248. B.1.1.248 has a number of S-proteinpolymorphisms [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y,H655Y, T1027I, V1176F] and is similar in certain key RBD positions(K417, E484, N501) to variant described from South Africa.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “B.1.1.28”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “B.1.1.248”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at positions 501 and 484 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, mRNA compositions and/ormethods described herein are characterized in that sera of vaccinatedsubjects display neutralizing activity against one or more SARs-CoV-2spike variants including a N501Y mutation and a E484K mutation in spikeprotein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 501 and 484 in spike protein as compared to SEQ IDNO: 1 or said one or more SARs-CoV-2 spike variants including a N501Ymutation and a E484K mutation in spike protein as compared to SEQ ID NO:1 may include one or more further mutations as compared to SEQ ID NO: 1(e.g., but not limited to H69/V70 deletion, Y144 deletion, A570D, D614G,P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S,D138Y, R190S, K417T, H655Y, T1027I, V1176F etc., as compared to SEQ IDNO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y and A701V as compared toSEQ ID NO: 1, and optionally: L18F, R246I, K417N, and deletion 242-244as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant may alsoinclude a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “B.1.1.248”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at positions 501, 484 and 614 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, mRNA compositions and/ormethods described herein are characterized in that sera of vaccinatedsubjects display neutralizing activity against one or more SARs-CoV-2spike variants including a N501Y mutation, a E484K mutation and a D614Gmutation in spike protein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 501, 484 and 614 in spike protein as compared toSEQ ID NO: 1 or said one or more SARs-CoV-2 spike variants including aN501Y mutation, a E484K mutation and a D614G mutation in spike proteinas compared to SEQ ID NO: 1 may include one or more further mutations ascompared to SEQ ID NO: 1 (e.g., but not limited to H69/V70 deletion,Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, A701V,L18F, R246I, K417N, L242/A243/L244 deletion, Y453F, I692V, S1147L,M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F etc., ascompared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a L242/A243/L244 deletion in spike protein as compared to SEQID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including aL242/A243/L244 deletion in spike protein as compared to SEQ ID NO: 1 mayinclude one or more further mutations as compared to SEQ ID NO: 1 (e.g.,but not limited to H69/V70 deletion, Y144 deletion, N501Y, A570D, D614G,P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I,K417N, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T,H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V and deletion242-244 as compared to SEQ ID NO: 1, and optionally: L18F, R246I, andK417N, as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at position 417 in spike protein as compared to SEQID NO: 1. In some embodiments, mRNA compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a K417N or K417T mutation in spike protein ascompared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 417 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a K417N or K417Tmutation in spike protein as compared to SEQ ID NO: 1 may include one ormore further mutations as compared to SEQ ID NO: 1 (e.g., but notlimited to H69/V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H,T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S,D138Y, R190S, H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V and K417N, ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “B.1.1.248”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereinare characterized in that sera of vaccinated subjects displayneutralizing activity against one or more SARs-CoV-2 spike variantsincluding a mutation at positions 417 and 484 and/or 501 in spikeprotein as compared to SEQ ID NO: 1. In some embodiments, mRNAcompositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a K417N or K417T mutation and aE484K and/or N501Y mutation in spike protein as compared to SEQ IDNO: 1. In some embodiments, one or more SARs-CoV-2 spike variantsincluding a mutation at positions 417 and 484 and/or 501 in spikeprotein as compared to SEQ ID NO: 1 or said one or more SARs-CoV-2 spikevariants including a K417N or K417T mutation and a E484K and/or N501Ymutation in spike protein as compared to SEQ ID NO: 1 may include one ormore further mutations as compared to SEQ ID NO: 1 (e.g., but notlimited to H69/V70 deletion, Y144 deletion, A570D, D614G, P681H, T716I,S982A, D1118H, D80A, D215G, A701V, L18F, R246I, L242/A243/L244 deletion,Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I,V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: D80A, D215G, E484K, N501Y, A701V and K417N, ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant “B.1.1.248”.

In particular embodiments, mRNA compositions and/or methods describedherein are characterized in that sera of vaccinated subjects displayneutralizing activity against SARs-CoV-2 spike variant including thefollowing mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

The SARs-CoV-2 spike variants described herein may or may not include aD614G mutation as compared to SEQ ID NO: 1.

In some embodiments, mRNA compositions and/or methods described hereincan provide protection against SARS-CoV-2 and/or decrease severity ofSARS-CoV-2 infection in at least 50% of subjects receiving such mRNAcompositions and/or methods.

In some embodiments, populations to be treated with mRNA compositionsdescribed herein include subjects of age 18-55. In some embodiments,populations to be treated with mRNA compositions described hereininclude subjects of age 56-85. In some embodiments, populations to betreated with mRNA compositions described herein include older subjects(e.g., over age 60, 65, 70, 75, 80, 85, etc, for example subjects of age65-85). In some embodiments, populations to be treated with mRNAcompositions described herein include subjects of age 18-85. In someembodiments, populations to be treated with mRNA compositions describedherein include subjects of age 18 or younger. In some embodiments,populations to be treated with mRNA compositions described hereininclude subjects of age 12 or younger. In some embodiments, populationsto be treated with mRNA compositions described herein include subjectsof age 10 or younger. In some embodiments, populations to be treatedwith mRNA compositions described herein may include adolescentpopulations (e.g., individuals approximately 12 to approximately 17years of age). In some embodiments, populations to be treated with mRNAcompositions described herein include infants (e.g., less than 1 yearold). In some embodiments, populations to be treated with mRNAcompositions described herein do not include infants (e.g., less than 1year) whose mothers have received such mRNA compositions describedherein during pregnancy. Without wishing to be bound by any particulartheory, a rat study as shown in Example 31 has suggested that aSARS-CoV-2 neutralizing antibody response induced in female rats givensuch mRNA compositions during pregnancy can pass onto fetuses. In someembodiments, populations to be treated with mRNA compositions describedherein include infants (e.g., less than 1 year) whose mothers did notreceive such mRNA compositions described herein during pregnancy. Insome embodiments, populations to be treated with mRNA compositionsdescribed herein may include pregnant women; in some embodiments,infants whose mothers were vaccinated during pregnancy (e.g., whoreceived at least one dose, or alternatively only who received bothdoses), are not vaccinated during the first weeks, months, or even years(e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 moths ormore, or 1, 2, 3, 4, 5 years or more) post-birth. Alternatively oradditionally, in some embodiments, infants whose mothers were vaccinatedduring pregnancy (e.g., who received at least one dose, or alternativelyonly who received both doses), receive reduced vaccination (e.g., lowerdoses and/or smaller numbers of administrations—e.g., boosters—and/orlower total exposure over a given period of time) after birth, forexample during the first weeks, months, or even years (e.g., 1, 2, 3, 4,5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3,4, 5 years or more) post-birthor may need reduced vaccination (e.g.,lower doses and/or smaller numbers of administrations—e.g.,boosters—over a given period of time), In some embodiments, compositionsas provided herein are administered to populations that do not includepregnant women.

In some particular embodiments, compositions as provided herein areadministered to pregnant women according to a regimen that includes afirst dose administered after about 24 weeks of gestation (e.g., afterabout 22, 23, 24, 25, 26, 27, 28 or more weeks of gestation); in someembodiments, compositions as provided herein are administered topregnant women according to a regimen that includes a first doseadministered before about 34 weeks of gestation (e.g., before about 30,31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In some embodiments,compositions as provided herein are administered to pregnant womenaccording to a regimen that includes a first dose administered afterabout 24 weeks (e.g., after about 27 weeks of gestation, e.g., betweenabout 24 weeks and 34 weeks, or between about 27 weeks and 34 weeks) ofgestation and a second dose administered about 21 days later; in someembodiments both doses are administered prior to delivery. Withoutwishing to be bound by any particular theory, it is proposed that such aregimen (e.g., involving administration of a first dose after about 24weeks, or 27 weeks of gestation and optionally before about 34 weeks ofgestation), and optionally a second dose within about 21 days, ideallybefore delivery, may have certain advantages in terms of safety (e.g.,reduced risk of premature delivery or of fetal morbidity or mortality)and/or efficacy (e.g., carryover vaccination imparted to the infant)relative to alternative dosing regimens (e.g., dosing at any time duringpregnancy, refraining from dosing during pregnancy, and/or dosing laterin pregnancy for example so that only one dose is administered duringgestation. In some embodiments, as noted herein (see also Example 34),infants born of mothers vaccinated during pregnancy, e.g, according to aparticular regimen as described herein, may not need furthervaccination, or may need reduced vaccination (e.g., lower doses and/orsmaller numbers of administrations—e.g., boosters—and/or lower overallexposure over a given period of time), for a period of time (e.g., asnoted herein) after birth.

In some embodiments, compositions as provided herein are administered topopulations in which women are advised against becoming pregnant for aperiod of time after receipt of the vaccine (e.g., after receipt of afirst dose of the vaccine, after receipt of a final dose of the vaccine,etc.); in some such embodiments, the period of time may be at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9weeks, at least 10 weeks or more, or may be at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, or more.

In some embodiments, populations to be treated with mRNA compositionsdescribed herein may include one or more populations with one or moreparticularly high risk conditions or history, e.g., as noted herein. Forexample, in some embodiments, populations to be treated with mRNAcompositions described herein may include subjects whose professionand/or environmental exposure may dramatically increase their risk ofgetting SARS-CoV-2 infection (including, e.g., but not limited to masstransportation, prisoners, grocery store workers, residents in long-termcare facilities, butchers or other meat processing workers, healthcareworkers, and/or first responders, e.g., emergency responders). Inparticular embodiments, populations to be treated with mRNA compositionsdescribed herein may include healthcare workers and/or first responders,e.g., emergency responders. In some embodiments, populations to betreated with mRNA compositions described herein may include those with ahistory of smoking or vaping (e.g., within 6 months, 12 months or more,including a history of chronic smoking or vaping). In some embodiments,populations to be treated with mRNA compositions described herein mayinclude certain ethnic groups that have been determined to be moresusceptible to SARS-CoV-2 infection.

In some embodiments, populations to be treated with mRNA compositionsdescribed herein may include certain populations with a blood type thatmay have been determined to more susceptible to SARS-CoV-2 infection. Insome embodiments, populations to be treated with mRNA compositionsdescribed herein may include immunocompromised subjects (e.g., thosewith HIV/AIDS; cancer and transplant patients who are taking certainimmunosuppressive drugs; autoimmune diseases or other physiologicalconditions expected to warrant immunosuppressive therapy (e.g., within 3months, within 6 months, or more); and those with inherited diseasesthat affect the immune system (e.g., congenital agammaglobulinemia,congenital IgA deficiency)). In some embodiments, populations to betreated with mRNA compositions described herein may include those withan infectious disease. For example, in some embodiments, populations tobe treated with mRNA compositions described herein may include thoseinfected with human immunodeficiency virus (HIV) and/or a hepatitisvirus (e.g., HBV, HCV). In some embodiments, populations to be treatedwith mRNA compositions described herein may include those withunderlying medical conditions. Examples of such underlying medicalconditions may include, but are not limited to hypertension,cardiovascular disease, diabetes, chronic respiratory disease, e.g.,chronic pulmonary disease, asthma, etc., cancer, and other chronicdiseases such as, e.g., lupus, rheumatoid arthritis, chronic liverdiseases, chronic kidney diseases (e.g., Stage 3 or worse such as insome embodiments as characterized by a glomerular filtration rate (GFR)of less than 60 mL/min/1.73 m²). In some embodiments, populations to betreated with mRNA compositions described herein may include overweightor obese subjects, e.g., specifically including those with a body massindex (BMI) above about 30 kg/m². In some embodiments, populations to betreated with mRNA compositions described herein may include subjects whohave prior diagnosis of COVID-19 or evidence of current or priorSARS-CoV-2 infection, e.g., based on serology or nasal swab. In someembodiments, populations to be treated include white and/ornon-Hispanic/non-Latino.

In some embodiments, certain mRNA compositions described herein (e.g.,BNT162b1) may be selected for administration to Asian populations (e.g.,Chinese populations), or in particular embodiments to older Asianpopulations (e.g, 60 years old or over, e.g., 60-85 or 65-85 years old).

In some embodiments, an mRNA composition as provided herein isadministered to and/or assessed in subject(s) who have been determinednot to show evidence of prior infection, and/or of present infection,before administration; in some embodiments, evidence of prior infectionand/or of present infection, may be or include evidence of intact virus,or any viral nucleic acid, protein, lipid etc. present in the subject(e.g., in a biological sample thereof, such as blood, cells, mucus,and/or tissue), and/or evidence of a subject's immune response to thesame. In some embodiments, an mRNA composition as provided herein isadministered to and/or assessed in subject(s) who have been determinedto show evidence of prior infection, and/or of present infection, beforeadministration; in some embodiments, evidence of prior infection and/orof present infection, may be or include evidence of intact virus, or anyviral nucleic acid, protein, lipid etc. present in the subject (e.g., ina biological sample thereof, such as blood, cells, mucus, and/ortissue), and/or evidence of a subject's immune response to the same. Insome embodiments, a subject is considered to have a prior infectionbased on having a positive N-binding antibody test result or positivenucleic acid amplification test (NAAT) result on the day of Dose 1.

In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject who has been informed of a risk of sideeffects that may include one or more of, for example: chills, fever,headache, injection site pain, muscle pain, tiredness; in someembodiments, an RNA (e.g., mRNA) composition is administered to asubject who has been invited to notify a healthcare provider if one ormore such side effects occurs, is experienced as more than mild ormoderate, persists for a period of more than a day or a few days, or ifany serious or unexpected event is experienced that the subjectreasonably considers may be associated with receipt of the composition.In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject who has been invited to notify a healthcareprovider of particular medical conditions which may include, forexample, one or more of allergies, bleeding disorder or taking a bloodthinner medication, breastfeeding, fever, immunocompromised state ortaking medication that affects the immune system, pregnancy or plan tobecome pregnant, etc. In some embodiments, an RNA (e.g., mRNA)composition as provided herein is administered to a subject who has beeninvited to notify a healthcare provider of having received anotherCOVID-19 vaccine. In some embodiments, an RNA (e.g., mRNA) compositionas provided herein is administered to a subject not having one of thefollowing medical conditions: experiencing febrile illness, receivingimmunosuppressant therapy, receiving anticoagulant therapy, sufferingfrom a bleeding disorder (e.g., one that would contraindicateintramuscular injection), or pregnancy and/or breastfeeding/lactation.In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject not having received another COVID-19vaccine. In some embodiments, an RNA (e.g., mRNA) composition asprovided herein is administered to a subject who has not had an allergicreaction to any component of the RNA (e.g., mRNA) composition. Examplesof such allergic reaction may include, but are not limited to difficultybreathing, swelling of fact and/or throat, fast hearbeat, rash,dizziness and/or weakness. In some embodiments, an RNA (e.g., mRNA)composition as provided herein is administered to a subject who receiveda first dose and did not have an allergic reaction (e.g., as describedherein) to the first dose. In some embodiments where allergic reactionoccurs in subject(s) after receiving a dose of an RNA (e.g., mRNA)composition as provided herein, such subject(s) may be administered oneor more interventions such as treatment to manage and/or reducesymptom(s) of such allergic reactions, for example, fever-reducingand/or anti-inflammatory agents.

In some embodiments, a subject who has received at least one dose of anRNA (e.g., mRNA) composition as provided herein is informed of avoidingbeing exposed to a coronavirus (e.g., SARS-CoV-2) unless and untilseveral days (e.g., at least 7 days, at least 8 days, 9 days, at least10 days, at least 11 days, at least 12 days, at least 13 days, at least14 days, etc.) have passed since administration of a second dose. Forexample, a subject who has received at at least one dose of an RNA(e.g., mRNA) composition as provided herein is informed of takingprecautionary measures against SARS-CoV-2 infection (e.g., remainingsocially distant, wearing masks, frequent hand-washing, etc.) unless anduntil several days (e.g., at least 7 days, at least 8 days, 9 days, atleast 10 days, at least 11 days, at least 12 days, at least 13 days, atleast 14 days, etc.) have passed since administration of a second dose.Accordingly, in some embodiments, methods of administering an RNA (e.g.,mRNA) composition as provided herein comprise administering a seconddose of such an RNA (e.g., mRNA) composition as provided herein to asubject who received a first dose and took precautionary measures toavoid being exposed to a coronavirus (e.g., SARS-CoV-2).

In some embodiments, mRNA compositions described herein may be deliveredto a draining lymph node of a subject in need thereof, for example, forvaccine priming. In some embodiments, such delivery may be performed byintramuscular administration of a provided mRNA composition.

In some embodiments, different particular mRNA compositions may beadministered to different subject population(s); alternatively oradditionally, in some embodiments, different dosing regimens may beadministered to different subject populations. For example, in someembodiments, mRNA compositions administered to particular subjectpopulation(s) may be characterized by one or more particular effects(e.g., incidence and/or degree of effect) in those subject populations.In some embodiments, such effect(s) may be or comprise, for exampletiter and/or persistence of neutralizing antibodies and/or T cells(e.g., T_(H)1-type T cells such as CD4⁺ and/or CD8⁺ T cells), protectionagainst challenge (e.g., via injection and/or nasal exposure, etc),incidence, severity, and/or persistence of side effects (e.g.,reactogenicity), etc.

In some embodiments, one or more mRNA compositions described herein maybe administered according to a regimen established to reduce COVID-19incidence per 1000 person-years, e.g., based on a laboratory test suchas nucleic acid amplification test (NAAT). In some embodiments, one ormore mRNA compositions described herein may be administered according toa regimen established to reduce COVID-19 incidence per 1000 person-yearsbased on a laboratory test such as nucleic acid amplification test(NAAT) in subjects receiving at least one dose of a provided mRNAcomposition with no serological or virological evidence (e.g., up to 7days after receipt of the last dose) of past SARS-CoV-2 infection. Insome embodiments, one or more mRNA compositions described herein may beadministered according to a regimen established to reduce confirmedsevere COVID-19 incidence per 1000 person-years. In some embodiments,one or more mRNA compositions described herein may be administeredaccording to a regimen established to reduce confirmed severe COVID-19incidence per 1000 person-years in subjects receiving at least one doseof a provided mRNA composition with no serological or virologicalevidence of past SARS-CoV-2 infection.

In some embodiments, one or more mRNA compositions described herein maybe administered according to a regimen established to produceneutralizing antibodies directed to a SARS-CoV-2 spike polypeptideand/or an immunogenic fragment thereof (e.g., RBD) as measured in serumfrom a subject that achieves or exceeds a reference level (e.g., areference level determined based on human SARS-CoV-2 infection/COVID-19convalescent sera) for a period of time and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) eptiopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD) for a period of time. In somesuch embodiments, the period of time may be at least 2 months, 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months or longer. In some embodiments, oneor more epitopes recognized by vaccine-induced T cells (e.g., CD8+ Tcells) may be presented on a MHC class I allele that is present in atleast 50% of subjects in a population, including, e.g., at least 60%, atleast 70%, at least 80%, at least 90%, or more; in some suchembodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402,HLA-B*3501, HLA-B*4401, or HLA-A*0201. In some embodiments, an epitopemay comprise HLA-A*0201 YLQPRTFLL (SEQ ID NO: 40); HLA-A*0201 RLQSLQTYV(SEQ ID NO: 41); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 42); HLA-A*2402NYNYLYRLF (SEQ ID NO: 43); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 44);HLA-B*3501 QPTESIVRF (SEQ ID NO: 45); HLA-B*3501 IPFAMQMAY (SEQ ID NO:46); or HLA-B*3501 LPFNDGVYF (SEQ ID NO: 47).

In some embodiments, efficacy is assessed as COVID-19 incidence per 1000person-years in individuals without serological or virological ecidenceof past SARS-CoV-2 infection before and during vaccination regimen;alternatively or additionally, in some embodiments, efficacy is assessedas COVID-19 incidence per 1000 person-years in subjects with and withoutevidence of past SARS-CoV-2 infection before and during vaccinationregimen. In some such embodiments, such incidence is of COVID-19 casesconfirmed within a specific time period after the final vaccination dose(e.g., a first dose in a single-dose regimen; a second dose in atwo-dose regimen, etc); in some embodiments, such time period may bewithin (i.e., up to and including 7 days) a particular number of days(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or more). In someembodiments, such time period may be within 7 days or within 14 days orwithin 21 days or within 28 days. In some embodiments, such time periodmay be within 7 days. In some embodiments, such time period may bewithin 14 days.

In some embodiments (e.g., in some embodiments of assessing efficacy), asubject is determined to have experienced COVID-19 infection if one ormore of the following is established: detection of SARS-CoV-2 nucleicacid in a sample from the subject, detection of antibodies thatspecifically recognize SARS-CoV-2 (e.g., a SARS-Co-V-2 spike protein),one or more symptoms of COVID-19 infection, and combinations thereof. Insome such embodiments, detection of SARS-CoV-2 nucleic acid may involve,for example, NAAT testing on a mid-turbinatae swap sample. In some suchembodiments, detection of relevant antibodies may involve serologicaltesting of a blood sample or portion thereof. In some such embodiments,symptoms of COVID-19 infection may be or include: fever, new orincreased cough, new or increased shortness of breath, chills, new orincreased muscle pain, new loss of taste or smell, sore throat,diarrhea, vomiting and combinations thereof. In some such embodiments,symptoms of COVID-19 infection may be or include: fever, new orincreased cough, new or increased shortness of breath, chills, new orincreased muscle pain, new loss of taste or smell, sore throat,diarrhea, vomiting, fatigue, headache, nasal congestion or runny nose,nausea, and combinations thereof. In some such embodiments, a subject isdetermined to have experienced COVID-19 infection if such subject bothhas experienced one such symptom and also has received a positive testfor SARS-CoV-2 nucleic acid or antibodies, or both. In some suchembodiments, a subject is determined to have experienced COVID-19infection if such subject both has experienced one such symptom and alsohas received a positive test for SARS-CoV-2 nucleic acid. In some suchembodiments, a subject is determined to have experienced COVID-19infection if such subject both has experienced one such symptom and alsohas received a positive test for SARS-CoV-2 antibodies.

In some embodiments (e.g., in some embodiments of assessing efficacy), asubject is determined to have experienced severe COVID-19 infection ifsuch subject has experienced one or more of: clinical signs at restindicative or severe systemic illness (e.g., one or more of respiratoryrate at greater than or equal to 30 breaths per minute, heart rate at orabove 125 beats per minute, SpO₂ less than or equal to 93% on room airat sea level or a PaO₂/FiO₂ below 300 m Hg), respiratory failure (e.g.,one or more of needing high-flow oxygen, noninvasive ventilation,mechanical ventilation, ECMO), evidence of shock (systolic bloodpressure below 90 mm Hg, diastolic blood pressure below 60 mm Hg,requiring vasopressors), significant acute renal, hepatic, or neurologicdystfunction, admission to an intensive care unit, death, andcombinations thereof.

In some embodiments, one or more mRNA compositions described herein maybe administered according to a regimen established to reduce thepercentage of subjects reporting at least one of the following: (i) oneor more local reactions (e.g., as described herein) for up to 7 daysfollowing each dose; (ii) one or more systemic events for up to 7 daysfollowing each dose; (iii) adverse events (e.g., as described herein)from a first dose to 1 month after the last dose; and/or (iv) seriousadverse events (e.g., as described herein) from a first dose to 6 monthsafter the last dose.

In some embodiments, one or more subjects who have received an RNA(e.g., mRNA) composition as described herein may be monitored (e.g., fora period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more,including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks ormore, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, including forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) to assess, forexample, presence of an immune response to component(s) of theadministered composition, evidence of exposure to and/or immune responseto SARS-CoV-2 or another coronavirus, evidence of any adverse event,etc. In some embodiments, monitoring may be via tele-visit.Alternatively or additionally, in some embodiments, monitoring may bein-person.

In some embodiments, a treatment effect conferred by one or more mRNAcompositions described herein may be characterized by (i) a SARS-CoV-2anti-S1 binding antibody level above a pre-determined threshold; (ii) aSARS-CoV-2 anti-RBD binding antibody level above a pre-determinedthreshold; and/or (iii) a SARS-CoV-2 serum neutralizing titer above athreshold level, e.g., at baseline, 1 month, 3 months, 6 months, 9months, 12 months, 18 months, and/or 24 months after completion ofvaccination. In some embodiments, anti-S1 binding antibody and/oranti-RBD binding antibody levels and/or serum neutralizing titers may becharacterized by geometric mean concentration (GMC), geometric meantiter (GMT), or geometric mean fold-rise (GMFR).

In some embodiments, a treatment effect conferred by one or more mRNAcompositions described herein may be characterized in that percentage oftreated subjects showing a SARS-CoV-2 serum neutralizing titer above apre-determined threshold, e.g., at baseline, 1 month, 3 months, 6months, 9 months, 12 months, 18 months, and/or 24 months aftercompletion of vaccination, is higher than the percentage of non-treatedsubjects showing a SARS-CoV-2 serum neutralizing titer above such apre-determined threshold (e.g., as described herein). In someembodiments, a serum neutralizing titer may be characterized bygeometric mean concentration (GMC), geometric mean titer (GMT), orgeometric mean fold-rise (GMFR).

In some embodiments, a treatment effect conferred by one or more mRNAcompositions described herein may be characterized by detection ofSARS-CoV-2 NVA-specific binding antibody.

In some embodiments, a treatment effect conferred by one or more mRNAcompositions described herein may be characterized by SARS-CoV-2detection by nucleic acid amplification test.

In some embodiments, a treatment effect conferred by one or more mRNAcompositions described herein may be characterized by induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) eptiopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD). In some embodiments, one ormore epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells)may be presented on a MHC class I allele that is present in at least 50%of subjects in a population, including, e.g., at least 60%, at least70%, at least 80%, at least 90%, or more; in some such embodiments, theMHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501,HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may compriseHLA-A*0201 YLQPRTFLL (SEQ ID NO: 40); HLA-A*0201 RLQSLQTYV (SEQ ID NO:41); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 42); HLA-A*2402 NYNYLYRLF (SEQ IDNO: 43); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 44); HLA-B*3501 QPTESIVRF(SEQ ID NO: 45); HLA-B*3501 IPFAMQMAY (SEQ ID NO: 46); or HLA-B*3501LPFNDGVYF (SEQ ID NO: 47).

In some embodiments, primary vaccine efficacy (VE) of one or more mRNAcompositions described herein may be established when there issufficient evidence (posterior probability) that either primary VE1 orboth primary VE1 and primary VE2 are >30% or higher (including, e.g.,greater than 40%, greater than 50%, greater than 60%, greater than 70%,greater than 80%, greater than 90%, greater than 95%, greater than 96%,greater than 97%, greater than 98%, or higher), wherein primary VE isdefined as primary VE=100×(1−IRR); and IRR is calculated as the ratio ofCOVID-19 illness rate in the vaccine group to the corresponding illnessrate in the placebo group. Primary VE1 represents VE for prophylacticmRNA compositions described herein against confirmed COVID-19 inparticipants without evidence of infection before vaccination, andprimary VE2 represents VE for prophylactic mRNA compositions describedherein against confirmed COVID-19 in all participants after vaccination.In some embodiments, primary VE1 and VE2 can be evaluated sequentiallyto control the overall type I error of 2.5% (hierarchical testing). Insome embodiments where one or more RNA (e.g., mRNA) compositionsdescribed herein are demonstrated to achieve primary VE endpoints asdiscussed above, secondary VE endpoints (e.g., confirmed severe COVID-19in participants without evidence of infection before vaccination andconfirmed severe COVID-19 in all participants) can be evaluatedsequentially, e.g., by the same method used for the primary VE endpointevaluation (hierarchical testing) as discussed above. In someembodiments, evaluation of primary and/or secondary VE endpoints may bebased on at least 20,000 or more subjects (e.g., at least 25,000 or moresubjects) randomized in a 1:1 ratio to the vaccine or placebo group,e.g., based on the following assumptions: (i) 1.0% illness rate per yearin the placebo group, and (ii) 20% of the participants beingnon-evaluable or having serological evidence of prior infection withSARS-CoV-2, potentially making them immune to further infection.

In some embodiments, one or more mRNA compositions described herein maybe administered according to a regimen established to achievemaintenance and/or continued enhancement of an immune response. Forexample, in some embodiments, an administration regimen may include afirst dose optionally followed by one or more subsequent doses; in someembodiments, need for, timing of, and/or magnitude of any suchsubsequent dose(s) may be selected to maintain, enhance, and/or modifyone or more immune responses or features thereof. In some embodiments,number, timing, and/or amount(s) of dose(s) have been established to beeffective when administered to a relevant population. In someembodiments, number, timing and/or amount(s) of dose(s) may be adjustedfor an individual subject; for example, in some embodiments, one or morefeatures of an immune response in an individual subject may be assessedat least once (and optionally more than once, for example multipletimes, typically spaced apart, often at pre-selected intervals) afterreceipt of a first dose. For example, presence of antibodies, B cells,and/or T cells (e.g., CD4⁺ and/or CD8⁺ T cells), and/or of cytokinessecreted thereby and/or identity of and/or extent of responses toparticular antigen(s) and/or epitope(s) may be assessed. In someembodiments, need for, timing of, and/or amount of a subsequent dose maybe determined in light of such assessments.

As noted hereinabove, in some embodiments, one or more subjects who havereceived an RNA (e.g., mRNA) composition as described herein may bemonitored (e.g., for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10days or more, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 weeks or more, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more,including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) fromreceipt of any particular dose to assess, for example, presence of animmune response to component(s) of the administered composition,evidence of exposure to and/or immune response to SARS-CoV-2 or anothercoronavirus, evidence of any adverse event, etc, including to performassessment of one or more of presence of antibodies, B cells, and/or Tcells (e.g., CD4⁺ and/or CD8⁺ T cells), and/or of cytokines secretedthereby and/or identity of and/or extent of responses to particularantigen(s) and/or epitope(s) may be assessed. Administration of acomposition as described herein may be in accordance with a regimen thatincludes one or more such monitoring steps.

For example, in some embodiments, need for, timing of, and/or amount ofa second dose relative to a first dose (and/or of a subsequent doserelative to a prior dose) is assessed, determined, and/or selected suchthat administration of such second (or subsequent) dose achievesamplification or modification of an immune response (e.g., as describedherein) observed after the first (or other prior) dose. In someembodiments, such amplification of an immune response (e.g., onesdescribed herein) may be at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, orhigher, as compared to the level of an immune response observed afterthe first dose. In some embodiments, such amplification of an immuneresponse may be at least 1.5 fold, at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, atleast 30-fold, or higher, as compared to the level of an immune responseobserved after the first dose.

In some embodiments, need for, timing of, and/or amount of a second (orsubsequent) dose relative to a first (or other prior) dose is assessed,determined, and/or selected such that administration of the later doseextends the durability of an immune response (e.g., as described herein)observed after the earlier dose; in some such embodiments, thedurability may be extended by at least 1 week, at least 2 weeks, atleast 3 weeks, at least 1 month, at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, or longer. In someembodiments, an immune response observed after the first dose may becharacterized by production of neutralizing antibodies directed to aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD) as measured in serum from a subject and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) eptiopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD). In some embodiments, one ormore epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells)may be presented on a MHC class I allele that is present in at least 50%of subjects in a population, including, e.g., at least 60%, at least70%, at least 80%, at least 90%, or more; in some such embodiments, theMHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501,HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may compriseHLA-A*0201 YLQPRTFLL (SEQ ID NO: 40); HLA-A*0201 RLQSLQTYV (SEQ ID NO:41); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 42); HLA-A*2402 NYNYLYRLF (SEQ IDNO: 43); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 44); HLA-B*3501 QPTESIVRF(SEQ ID NO: 45); HLA-B*3501 IPFAMQMAY (SEQ ID NO: 46); or HLA-B*3501LPFNDGVYF (SEQ ID NO: 47).

In some embodiments, need for, timing of, and/or amount of a second doserelative to a first dose (or other subsequent dose relative to a priordose) is assessed, determined and/or selected such that administrationof such second (or subsequent) dose maintains or exceeds a referencelevel of an immune response; in some such embodiments, the referencelevel is determined based on human SARS-CoV-2 infection/COVID-19convalescent sera and/or PBMC samples drawn from subjects (e.g., atleast a period of time such as at least 14 days or longer, including,e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, or longer,after PCR-confirmed diagnosis when the subjects were asymptomatic. Insome embodiments, an immune response may be characterized by productionof neutralizing antibodies directed to a SARS-CoV-2 spike polypeptideand/or an immunogenic fragment thereof (e.g., RBD) as measured in serumfrom a subject and/or induction of cell-mediated immune response (e.g.,a T cell response against SARS-CoV-2), including, e.g., in someembodiments induction of T cells that recognize at least one or moreMHC-restricted (e.g., MHC class I-restricted) eptiopes within aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD). In some embodiments, one or more epitopes recognized byvaccine-induced T cells (e.g., CD8+ T cells) may be presented on a MHCclass I allele that is present in at least 50% of subjects in apopulation, including, e.g., at least 60%, at least 70%, at least 80%,at least 90%, or more; in some such embodiments, the MHC class I allelemay be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. Insome embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL (SEQ IDNO: 40); HLA-A*0201 RLQSLQTYV (SEQ ID NO: 41); HLA-A*2402 QYIKWPWYI (SEQID NO: 42); HLA-A*2402 NYNYLYRLF (SEQ ID NO: 43); HLA-A*2402 KWPWYIWLGF(SEQ ID NO: 44); HLA-B*3501 QPTESIVRF (SEQ ID NO: 45); HLA-B*3501IPFAMQMAY (SEQ ID NO: 46); or HLA-B*3501 LPFNDGVYF (SEQ ID NO: 47).

In some embodiments, determination of need for, timing of, and/or amountof a second (or subsequent) dose may include one or more steps ofassessing, after (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21 days or longer after) a first (or other prior) dose, presenceand/or expression levels of neutralizing antibodies directed to aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD) as measured in serum from a subject and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) eptiopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD). In some embodiments, one ormore epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells)may be presented on a MHC class I allele that is present in at least 50%of subjects in a population, including, e.g., at least 60%, at least70%, at least 80%, at least 90%, or more; in some such embodiments, theMHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501,HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may compriseHLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI;HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF;HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

In some embodiments, a kit as provided herein may comprise a real-timemonitoring logging device, which, for example in some embodiments, iscapable of providing shipment temperatures, shipment time and/orlocation.

In some embodiments, an RNA (e.g., mRNA) composition as described hereinmay be shipped, stored, and/or utilized, in a container (such as a vialor syringe), e.g., a glass container (such as a glass vial or syringe),which, in some embodiments, may be a single-dose container or amulti-dose container (e.g., may be arranged and constructed to hold,and/or in some embodiments may hold, a single dose, or multiple doses ofa product for administration). In some embodiments, a multi-dosecontainer (such as a multi-dose vial or syringe) may be arranged andconstructed to hold, and/or may hold 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredoses; in some particular embodiments, it may be designed to hold and/ormay hold 5 doses. In some embodiments, a single-dose or multi-dosecontainer (such as a single-dose or multi-dose vial or syringe) may bearranged and constructed to hold and/or may hold a volume or amountgreater than the indicated number of doses, e.g., in order to permitsome loss in transfer and/or administration. In some embodiments, an RNA(e.g., mRNA) composition as described herein may be shipped, stored,and/or utilized, in a preservative-free glass container (e.g., apreservative-free glass vial or syringe, e.g., a single-dose ormulti-dose preservative-free glass vial or syringe). In someembodiments, an RNA (e.g., mRNA) composition as described herein may beshipped, stored, and/or utilized, in a preservative-free glass container(e.g., a preservative-free glass vial or syringe, e.g., a single-dose ormulti-dose preservative-free glass vial or syringe) that contains 0.45ml of frozen liquid (e.g., including 5 doses). In some embodiments, anRNA (e.g., mRNA) composition as described herein and/or a container(e.g., a vial or syringe) in which it is disposed, is shipped, stored,and/or utilized may be maintained at a temperature below roomtemperature, at or below 4° C., at or below 0° C., at or below −20° C.,at or below −60° C., at or below −70° C., at or below −80° C., at orbelow −90° C., etc. In some embodiments, an RNA (e.g., mRNA) compositionas described herein and/or a container (e.g., a viral or syringe) inwhich it is disposed, is shipped, stored, and/or utilized may bemaintained at a temperature between −80° C. and −60° C. and in someembodiments protected from light. In some embodiments, an RNA (e.g.,mRNA) composition as described herein and/or a container (e.g., a viralor syringe) in which it is disposed, is shipped, stored, and/or utilizedmay be maintained at a temperature below about 25° C., and in someembodiments protected from light. In some embodiments, an RNA (e.g.,mRNA) composition as described herein and/or a container (e.g., a viralor syringe) in which it is disposed, is shipped, stored, and/or utilizedmay be maintained at a temperature below about 5° C. (e.g., below about4° C.), and in some embodiments protected from light. In someembodiments, an RNA (e.g., mRNA) composition as described herein and/ora container (e.g., a viral or syringe) in which it is disposed, isshipped, stored, and/or utilized may be maintained at a temperaturebelow about −20° C., and in some embodiments protected from light. Insome embodiments, an RNA (e.g., mRNA) composition as described hereinand/or a container (e.g., a viral or syringe) in which it is disposed,is shipped, stored, and/or utilized may be maintained at a temperatureabove about −60° C. (e.g., in some embodiments at or above about −20°C., and in some embodiments at or above about 4-5° C., in either caseoptionally below about 25° C.), and in some embodiments protected fromlight, or otherwise without affirmative steps (e.g., cooling measures)taken to achieve a storage temperature materially below about −20° C.

In some embodiments, an RNA (e.g., mRNA) composition as described hereinand/or a container (e.g., a vial or syringe) in which it is disposed isshipped, stored, and/or utilized together with and/or in the context ofa thermally protective material or container and/or of a temperatureadjusting material. For example, in some embodiments, an RNA (e.g.,mRNA) composition as described herein and/or a container (e.g., a vialor syringe) in which it is disposed is shipped, stored, and/or utilizedtogether with ice and/or dry ice and/or with an insulating material. Insome particular embodiments, a container (e.g., a vial or syringe) inwhich an RNA (e.g., mRNA) composition is disposed is positioned in atray or other retaining device and is further contacted with (orotherwise in the presence of) temperature adjusting (e.g., ice and/ordry ice) material and/or insulating material. In some embodiments,multiple containers (e.g., multiple vials or syringes such as single useor multi-use vials or syringes as described herein) in which a providedRNA (e.g., mRNA) composition is disposed are co-localized (e.g., in acommon tray, rack, box, etc.) and packaged with (or otherwise in thepresence of) temperature adjusting (e.g., ice and/or dry ice) materialand/or insulating material. To give but one example, in someembodiments, multiple containers (e.g., multiple vials or syringes suchas single use or multi-use vials or syringes as described herein) inwhich an RNA (e.g., mRNA) composition is disposed are positioned in acommon tray or rack, and multiple such trays or racks are stacked in acarton that is surrounded by a temperature adjusting material (e.g., dryice) in a thermal (e.g., insulated) shipper. In some embodiments,temperature adjusting material is replenished periodically (e.g., within24 hours of arrival at a site, and/or every 2 hours, 4 hours, 6 hours, 8hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, etc.). Preferably, re-entry into a thermal shipper shouldbe infrequent, and desirably should not occur more than twice a day. Insome embodiments, a thermal shipper is re-closed within 5, 4, 3, 2, or 1minute, or less, of having been opened. In some embodiments, a providedRNA (e.g., mRNA) composition that has been stored within a thermalshipper for a period of time, optionally within a particular temperaturerange remains useful. For example, in some embodiments, if a thermalshipper as described herein containing a provided RNA (e.g., mRNA)composition is or has been maintained (e.g., stored) at a temperaturewithin a range of about 15° C. to about 25° C., the RNA (e.g., mRNA)composition may be used for up to 10 days; that is, in some embodiments,a provided RNA (e.g., mRNA) composition that has been maintained withina thermal shipper, which thermal shipper is at a temperature within arange of about 15° C. to about 25° C., for a period of not more than 10days is administered to a subject. Alternatively or additionally, insome embodiments, if a provided RNA (e.g., mRNA) composition is or hasbeen maintained (e.g., stored) within a thermal shipper, which thermalshipper has been maintained (e.g., stored) at a temperature within arange of about 15° C. to about 25° C., it may be used for up to 10 days;that is, in some embodiments, a provided RNA (e.g., mRNA) compositionthat has been maintained within a thermal shipper, which thermal shipperhas been maintained at a temperature within a range of about 15° C. toabout 25° C. for a period of not more than 10 days is administered to asubject.

In some embodiments, a provided RNA (e.g., mRNA) composition is shippedand/or stored in a frozen state. In some embodiments, a provided RNA(e.g., mRNA composition is shipped and/or stored as a frozen suspension,which in some embodiments does not contain preservative. In someembodiments, a frozen RNA (e.g., mRNA) composition is thawed. In someembodiments, a thawed RNA (e.g., mRNA) composition (e.g., a suspension)may contain white to off-white opaque amorphous particles. In someembodiments, a thawed RNA (e.g., mRNA) composition may be used for up toa small number (e.g., 1, 2, 3, 4, 5, or 6) of days after thawing ifmaintained (e.g., stored) at a temperature at or below room temperature(e.g., below about 30° C., 25° C., 20° C., 15° C., 10° C., 8° C., 4° C.,etc). In some embodiments, a thawed RNA (e.g., mRNA) composition may beused after being stored (e.g., for such small number of days) at atemperature between about 2° C. and about 8° C.; alternatively oradditionally, a thawed RNA (e.g., mRNA) composition may be used within asmall number (e.g., 1, 2, 3, 4, 5, 6) of hours after thawing at roomtemperature. Thus, in some embodiments, a provided RNA (e.g., mRNA)composition that has been thawed and maintained at a temperature at orbelow room temperature, and in some embodiments between about 2° C. andabout 8° C., for not more than 6, 5, 4, 3, 2, or 1 days is administeredto a subject. Alternatively or additionally, in some embodiments, aprovided RNA (e.g., mRNA) composition that has been thawed andmaintained at room temperature for not more than 6, 5, 4, 3, 2, or 1hours is administered to a subject. In some embodiments, a provided RNA(e.g., mRNA) composition is shipped and/or stored in a concentratedstate. In some embodiments, such a concentrated composition is dilutedprior to administration. In some embodiments, a diluted composition isadministered within a period of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1hour(s) post-dilution; in some embodiments, such administration iswithin 6 hours post-dilution. Thus, in some embodiments, dilutedpreparation of a provided RNA (e.g., mRNA) composition is administeredto a subject within 6 hours post-dilution (e.g., as described hereinafter having been maintained at an appropriate temperature, e.g., at atemperature below room temperature, at or below 4° C., at or below 0°C., at or below −20° C., at or below −60° C., at or below −70° C., at orbelow −80° C., etc, and typically at or above about 2° C., for examplebetween about 2° C. and about 8° C. or between about 2° C. and about 25°C.). In some embodiments, unusued composition is discarded withinseveral hours (e.g., about 10, about 9, about 8, about 7, about 6, about5 or fewer hours) after dilution; in some embodiments, unusedcomposition is discarded within 6 hours of dilution.

In some embodiments, an RNA (e.g., mRNA) composition that is stored,shipped or utilized (e.g., a frozen composition, a liquid concentratedcomposition, a diluted liquid composition, etc.) may have beenmaintained at a temperature materially above −60° C. for a period oftime of at least 1, 2, 3, 4, 5, 6, 7 days or more, or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 weeks or more, or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 months or more; in some such embodiments, such compositionmay have been maintained at a temperature at or above about −20° C. forsuch period of time, and/or at a temperature up to or about 4-5° C. forsuch period of time, and/or may have been maintained at a temperatureabove about 4-5° C., and optionally about 25° C. for a period of time upthat is less than two (2) months and/or optionally up to about one (1)month. In some embodiments, such composition may not have been stored,shipped or utilized (or otherwise exposed to) a temperature materiallyabove about 4-5° C., and in particular not at or near a temperature ofabout 25° C. for a period of time as long as about 2 weeks, or in someembodiments 1 week. In some embodiments, such composition may not havebeen stored, shipped or utilized (or otherwise exposed to) a temperaturematerially above about −20° C., and in particular not at or near atemperature of about 4-5° C. for a period of time as long as about 12months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5months, 4 months, 3 months, 2 months, or, in some embodiments, for aperiod of time as long as about 8 weeks or 6 weeks or materially morethan about 2 months or, in some embodiments, 3 months or, in someembodiments 4 months.

In some embodiments, an RNA (e.g., mRNA) composition that is stored,shipped or utilized (e.g., a frozen composition, a liquid concentratedcomposition, a diluted liquid composition, etc.) may be protected fromlight. In some embodiments, one or more steps may be taken to reduce orminimize exposure to light for such compositions (e.g., which may bedisposed within a container such as a vial or a syringe). In someembodiments, exposure to direct sunlight and/or to ultraviolent light isavoided. In some embodiments, a diluted solution may be handled and/orutilized under normal room light conditions (e.g., without particularsteps taken to minimize or reduce exposure to room light). It should beunderstood that strict adherence to aseptic techniques is desirableduring handling (e.g., diluting and/or administration) of an RNA (e.g.,mRNA) composition as described herein. In some embodiments, an RNA(e.g., mRNA) composition as described herein is not administered (e.g.,is not injected) intravenously. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) intradermally. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) subcutaneously. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) any of intravenously, intradermally, or subcutaneously. Insome embodiments, an RNA (e.g., mRNA) composition as described herein isnot administered to a subject with a known hypersensitivity to anyingredient thereof. In some embodiments, a subject to whom an RNA (e.g.,mRNA) composition has been administered is monitored for one or moresigns of anaphylaxis. In some embodiments, a subject to whom an RNA(e.g., mRNA) composition is administered had previously received atleast one dose of a different vaccine for SARS-CoV-2; in someembodiments, a subject to whom an RNA (e.g., mRNA) composition isadministered had not previously received a different vaccine forSARS-CoV-2. In some embodiments, a subject's temperature is takenpromptly prior to administration of an RNA (e.g., mRNA) composition(e.g., shortly before or after thawing, dilution, and/or administrationof such composition); in some embodiments, if such subject is determinedto be febrile, administration is delayed or canceled. In someembodiments, an RNA (e.g., mRNA) composition as described herein is notadministered to a subject who is receiving anticoagulant therapy or issuffering from or susceptible to a bleeding disorder or condition thatwould contraindicate intramuscular injection. In some embodiments, anRNA (e.g., mRNA) composition as described herein is administered by ahealthcare professional who has communicated with the subject receivingthe composition information relating to side effects and risks. In someembodiments, an RNA (e.g., mRNA) composition as described herein isadministered by a healthcare professional who has agreed to submit anadverse event report for any serious adverse events, which may includefor example one or more of death, development of a disability orcongenital anomaly/birth defect (e.g., in a child of the subject),in-patient hospitalization (including prolongation of an existinghospitalization), a life-threatening event, a medical or surgicalintervention to prevent death, a persistent or significant orsubstantial disruption of the ability to conduct normal life functions;or another important medical event that may jeopardize the individualand may require medical or surgical intervention (treatment) to preventone of the other outcomes. In some embodiments, provided RNAcompositions are administered to a population of individuals under 18years of age, or under 17 years of age, or under 16 years of age, orunder 15 years of age, or under 14 years of age, or under 13 years ofage, for example according to a regimen established to have a rate ofincidence for one or more of the local reaction events indicated belowthat does not exceed the rate of incidence indicated below:

-   -   pain at the injection site (75% after a first dose and/or a        second dose, and/or a lower incidence after a second dose, e.g.,        65% after a second dose);    -   redness at the injection site (less than 5% after a first dose        and/or a second dose); and/or    -   swelling at the injection site (less than 5% after a first dose        and/or a second dose).

In some embodiments, provided RNA compositions are administered to apopulation of individuals under 18 years of age, or under 17 years ofage, or under 16 years of age, or under 15 years of age, or under 14years of age, or under 13 years of age, for example according to aregimen established to have a rate of incidence for one or more of thesystemic reaction events indicated below that does not exceed the rateof incidence indicated below:

-   -   fatigue (55% after a first dose and/or a second dose);    -   headache (50% after a first dose and/or a second dose);    -   muscle pain (40% after a first dose and/or a second dose);    -   chills (40% after a first dose and/or a second dose);    -   joint pain (20% after a first dose and/or a second dose);    -   fever (25% after a first dose and/or a second dose);    -   vomiting (10% after a first dose and/or a second dose); and/or    -   diarrhea (10% after a first dose and/or a second dose).

In some embodiments, medication that alleviates one or more symptoms ofone or more local reaction and/or systemic reaction events (e.g.,described herein) are administered to individuals under 18 years of age,or under 17 years of age, or under 16 years of age, or under 15 years ofage, or under 14 years of age, or under 13 years of age who have beenadministered with provided RNA compositions and have experienced one ormore of the local and/or systemic reaction events (e.g., describedherein). In some embodiments, antipyretic and/or pain medication can beadministered to such individuals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Schematic overview of the S protein organization of theSARS-CoV-2 S protein.

The sequence within the S1 subunit consists of the signal sequence (SS)and the receptor binding domain (RBD) which is the key subunit withinthe S protein which is relevant for binding to the human cellularreceptor ACE2. The S2 subunit contains the S2 protease cleavage site(S2′) followed by a fusion peptide (FP) for membrane fusion, heptadrepeats (HR1 and HR2) with a central helix (CH) domain, thetransmembrane domain (TM) and a cytoplasmic tail (CT).

FIG. 2 : Anticipated constructs for the development of a SARS-CoV-2vaccine.

Based on the full and wildtype S protein, we have designed differentconstruct encoding the (1) full protein with mutations in close distanceto the first heptad repeat (HRP1) that include stabilizing mutationspreserving neutralisation sensitive sites, the (2) S1 domain or the (3)RB domain (RBD) only. Furthermore, to stabilize the protein fragments afibritin domain (F) was fused to the C-terminus. All constructs startwith the signal peptide (SP) to ensure Golgi transport to the cellmembrane.

FIG. 3 : Antibody immune response against Influenza HA using theLNP-formulated modRNA.

BALB/c mice were immunized twice with 1 μg of the vaccine candidate.Total amount of viral antigen specific immunoglobulin G (IgG) wasmeasured via ELISA. The functionality of the antibodies was assessed viaVNT.

FIG. 4 : T cell response against Influenza HA using the LNP-formulatedmodRNA platform.

BALB/c mice were immunized IM with 1 μg of the vaccine candidate, twice.The T cell response was analyzed using antigen specific peptides for Tcell stimulation recovered from the spleen. IFNγ release was measuredafter peptide stimulation using an ELISpot assay.

FIG. 5 : Anti-S protein IgG response 7, 14, 21 and 28 d afterimmunization with BNT162a1.

BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulatedRBL063.3. On day 7, 14, 21 and 28 after immunization, animals were bledand the serum samples were analyzed for total amount of anti-S1 (left)and anti-RBD (right) antigen specific immunoglobulin G (IgG) measuredvia ELISA. For day 7, day 14, day 21 and day 28, values for a serumdilution of 1:100 were included in the graph. One point in the graphstands for one mouse, every mouse sample was measured in duplicates(group size n=8; mean+SEM is included for the groups).

FIG. 6 : Anti-S protein IgG response 7, 14, 21 and 28 d afterimmunization with BNT162b1.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBP020.3. On day 7, 14, 21 and 28 after immunization, animals were bledand the serum samples were analyzed for total amount of anti-S1 (left)and anti-RBD (right) antigen specific immunoglobulin G (IgG) measuredvia ELISA. For day 7 (1:100), day 14 (1:300), day 21 (1:900), and day 28(1:2700) different serum dilution were included in the graph. One pointin the graph stands for one mouse, every mouse sample was measured induplicates (group size n=8; mean+SEM is included for the groups).

FIG. 7 : Neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d afterimmunization with BNT162b1.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBP020.3. On 14, 21 and 28 d after immunization, animals were bled, andthe sera were tested for SARS CoV-2 pseudovirus neutralization. Graphsdepict pVN50 serum dilutions (50% reduction of infectious events,compared to positive controls without serum). One point in the graphsstands for one mouse. Every mouse sample was measured in duplicate.Group size n=8. Mean+SEM is shown by horizontal bars with whiskers foreach group. LLOQ, lower limit of quantification. ULOQ, upper limit ofquantification.

FIG. 8 : Anti-S protein IgG response 7, 14 and 21 d after immunizationwith BNT162c1.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBS004.3. On day 7, 14 and 21 after immunization, animals were bled andthe serum samples were analyzed for total amount of anti-S1 (left) andanti-RBD (right) antigen specific immunoglobulin G (IgG) measured viaELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) differentserum dilution were included in the graph. One point in the graph standsfor one mouse, every mouse sample was measured in duplicates (group sizen=8; mean+SEM is included for the groups).

FIG. 9 : Neutralization of SARS-CoV-2 pseudovirus 14 and 21 d afterimmunization with BNT162c1.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBS004.3. On 14 and 21 d after immunization, animals were bled and thesera were tested for SARS CoV-2 pseudovirus neutralization. Graphsdepict pVN50 serum dilutions (50% reduction of infectious events,compared to positive controls without serum). One point in the graphsstands for one mouse. Every mouse sample was measured in duplicate.Group size n=8. Mean+SEM is shown by horizontal bars with whiskers foreach group. LLOQ, lower limit of quantification. ULOQ, upper limit ofquantification.

FIG. 10 : Anti-S protein IgG response 7, 14, 21 and 28 d afterimmunization with LNP-formulated RBL063.1.

BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulatedRBL063.1. On day 7, 14, 21 and 28 after immunization, animals were bledand the serum samples were analyzed for total amount of anti-S1 (left)and anti-RBD (right) antigen specific immunoglobulin G (IgG) measuredvia ELISA. For day 7 (1:100), day 14 (1:100), day 21 (1:300) and day 28(1:900) different serum dilution were included in the graph. One pointin the graph stands for one mouse, every mouse sample was measured induplicates (group size n=8; mean+SEM is included for the groups).

FIG. 11 : Neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d afterimmunization with LNP-formulated RBL063.1.

BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulatedRBL063.1. On 14, 21, and 28 d after immunization, animals were bled andthe sera were tested for SARS CoV-2 pseudovirus neutralization. Graphsdepict pVN50 serum dilutions (50% reduction of infectious events,compared to positive controls without serum). One point in the graphsstands for one mouse. Every mouse sample was measured in duplicate.Group size n=8. Mean+SEM is shown by horizontal bars with whiskers foreach group. LLOQ, lower limit of quantification. ULOQ, upper limit ofquantification.

FIG. 12 : Anti-S protein IgG response 7, 14 and 21 d after immunizationwith BNT162b2 (LNP-formulated RBP020.1).

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg ofLNP-formulatedRBP020.1. On day 7, 14, and 21 after immunization, animalswere bled and the serum samples were analyzed for total amount ofanti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G(IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21(1:1100) different serum dilution were included in the graph. One pointin the graph stands for one mouse, every mouse sample was measured induplicates (group size n=8; mean+SEM is included for the groups).

FIG. 13 : Neutralization of SARS-CoV-2 pseudovirus 14 and 21 afterimmunization with BNT162b2 (LNP-formulated RBP020.1).

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBP020.1. On day 14 and 21 after immunization, animals were bled and thesera were tested for SARS CoV-2 pseudovirus neutralization. Graphsdepict pVN50 serum dilutions (50% reduction of infectious events,compared to positive controls without serum). One point in the graphsstands for one mouse. Every mouse sample was measured in duplicate.Group size n=8. Mean+SEM is shown by horizontal bars with whiskers foreach group. LLOQ, lower limit of quantification. ULOQ, upper limit ofquantification.

FIG. 14 : Anti-S protein IgG response 7, 14 and 21 d after immunizationwith LNP-formulated RBS004.2.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBS004.2. On day 7, 14 and 21 after immunization, animals were bled andthe serum samples were analyzed for total amount of anti-S1 (left) andanti-RBD (right) antigen specific immunoglobulin G (IgG) measured viaELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) differentserum dilution were included in the graph. One point in the graph standsfor one mouse, every mouse sample was measured in duplicates (group sizen=8; mean+SEM is included for the groups).

FIG. 15 : Neutralization of SARS-CoV-2 pseudovirus 14 and 21 afterimmunization with LNP-formulated RBS004.2.

BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulatedRBS004.2. On 14, and 21 d after immunization, animals were bled, and thesera were tested for SARS CoV-2 pseudovirus neutralization. Graphsdepict pVN50 serum dilutions (50% reduction of infectious events,compared to positive controls without serum). One point in the graphsstands for one mouse. Every mouse sample was measured in duplicate.Group size n=8. Mean+SEM is shown by horizontal bars with whiskers foreach group. LLOQ, lower limit of quantification. ULOQ, upper limit ofquantification.

FIG. 16 : ALC-0315 activity in the screening process.

FIG. 17 : Luciferase expression was monitored on the right (site ofinjection), dorsal (site of injection) and ventral (drainage to theliver) sides of the animal after intramuscular administration inwild-type (WT) or ApoE knockout C57Bl/6 mice in the presence or absenceof ApoE3. Luciferase expression was detected using Xenolight D-LuciferinRediject at 4, 24, 72 and 96 hours post administration.

FIG. 18 : Luciferase activity after intravenous (IV) and intramuscular(IM) administration in wild-type (WT) or ApoE knockout C57Bl/6 mice inthe presence (KO+) or absence (KO) of ApoE3. Luciferase expression wasdetected using Xenolight D-Luciferin Rediject at 4 hours postadministration.

FIG. 19 : General structure of the RNA.

Schematic illustration of the general structure of the RNA vaccines with5′-cap, 5′- and 3′-untranslated regions, coding sequences with intrinsicsecretory signal peptide as well as GS-linker, and poly(A)-tail. Pleasenote that the individual elements are not drawn exactly true to scalecompared to their respective sequence lengths.

UTR=Untranslated region; sec=Secretory signal peptide; RBD=ReceptorBinding Domain; GS=Glycine-serine linker.

FIG. 20 : General structure of the RNA.

Schematic illustration of the general structure of the RNA drugsubstances with 5′-cap, 5′- and 3′-untranslated regions, codingsequences with intrinsic secretory signal peptide as well as GS-linker,and poly(A)-tail. Please note that the individual elements are not drawnexactly true to scale compared to their respective sequence lengths.

GS=Glycine-serine linker; UTR=Untranslated region; Sec=Secretory signalpeptide; RBD=Receptor Binding Domain.

FIG. 21 : General structure of the RNA.

Schematic illustration of the general structure of the RNA vaccines with5′-cap, 5′- and 3′-untranslated regions, coding sequences of theVenezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerasereplicase and the SARS-CoV-2 antigen with intrinsic secretory signalpeptide as well as GS-linker, and poly(A)-tail. Please note that theindividual elements are not drawn exactly true to scale compared totheir respective sequence lengths. UTR=Untranslated region;Sec=Secretory signal peptide; RBD=Receptor Binding Domain;GS=Glycine-serine linker.

FIG. 22 : ELISpot analysis 28 d after immunization with BNT162b1.

BALB/c mice were immunized IM once with 1 μg of LNP-formulated RBP020.3.On day 28 after immunization, mice were euthanized and splenocytes wereprepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ Tcells. T cells were stimulated with an S protein- or RBD-specificoverlapping peptide pool and IFN-γ secretion was measured to assessT-cell responses. One point in the graph stands for the individual spotcount of one mouse, every mouse sample was measured in duplicates (groupsize n=8; mean is included for the groups).

FIG. 23 : Cytokine concentrations in supernatants of re-stimulatedsplenocytes 12 d after immunization with BNT162b1.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3.On day 12 after immunization, mice were euthanized. Splenocytes wereprepared and were stimulated with an S protein-specific overlappingpeptide pool. After 48 h of stimulation, supernatant was collected andcytokine concentrations were determined. One point in the graph standsfor the individual cytokine concentration of one mouse, every mousesample was measured in duplicates (group size n=8; mean is included forthe groups).

FIG. 24 : T cell immunophenotyping in PBMCs 7 days after immunizationwith BNT162b1.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3.On day 7 after immunization, mice were bled. Flow cytometry analysis ofPBMCs was performed of T cells. T cells were defined as viable CD3⁺CD4⁺and CD3⁺CD8⁺ T cells. Additional phenotyping markers are included in thefigures. Tfh cells were gated from CD4⁺ T cells and defined as CD4⁺T-bet-GATA3⁻CD44⁺CD62L⁻PD−1⁺CXCR5⁺ cells. One point in the graph standsfor the individual cell fraction of one mouse (group size n=8; mean isincluded for the groups).

FIG. 25 : B cell immunophenotyping in draining lymph nodes 12 days afterimmunization with BNT162b1.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3.On day 12 after immunization, mice were euthanized. Flow cytometryanalysis of lymphocytes was performed of B cells. Activated B cells weregated within single, viable lymphocytes and defined as IgD-Dump (CD4,CD8, F4/80, GR-1)⁻ cells. Plasma cells were defined asCD138⁺B220^(low/−) cells. Switched B cells were gated from non-plasmacells and defined as CD19⁺CD138⁻IgM⁻. Germinal center (GC) B cells weregated from switched B cells and defined as CD19⁺IgM⁻CD38⁻CD95⁺ cells andgated for IgG1 and IgG2a. One point in the graph stands for theindividual cell fraction of one mouse (group size n=8; mean is includedfor the groups).

FIG. 26 : ELISpot analysis 28 d after immunization with LNP-formulatedmodRNA RBP020.1.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBP020.1.On day 28 after immunization, mice were euthanized and splenocytes wereprepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ Tcells. T cells were stimulated with an S protein-specific overlappingpeptide pool and IFN-γ secretion was measured to assess T-cellresponses. One point in the graph stands for the individual spot countof one mouse, every mouse sample was measured in duplicates (group sizen=8; mean is included for the groups).

FIG. 27 : Cytokine concentrations in supernatants of re-stimulatedsplenocytes 28 d after immunization with LNP-formulated modRNA RBP020.1.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBP020.1.On day 28 after immunization, mice were euthanized. Splenocytes wereprepared and were stimulated with an S protein-specific overlappingpeptide pool. After 48 h of stimulation, supernatant was collected andcytokine concentrations were determined. One point in the graph standsfor the individual cytokine concentration of one mouse, every mousesample was measured in duplicates (group size n=8; mean is included forthe groups).

FIG. 28 : ELISpot analysis 28 d after immunization with LNP-formulatedsaRNA RBS004.2.

BALB/c mice were immunized IM once with 5 μg of LNP-formulated RBS004.2.On day 28 after immunization, mice were euthanized and splenocytes wereprepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ Tcells. T cells were stimulated with an S protein-specific overlappingpeptide pool and IFN-γ secretion was measured to assess T-cellresponses. One point in the graph stands for the individual spot countof one mouse, every mouse sample was measured in duplicates (group sizen=8; mean is included for the groups).

FIG. 29 : Cytokine concentrations in supernatants of re-stimulatedsplenocytes 28 d after immunization with LNP-formulated saRNA RBS004.2.

BALB/c mice were immunized IM once with 1 μg of LNP-formulated RBS004.2.On day 28 after immunization, mice were euthanized. Splenocytes wereprepared and were stimulated with an S protein-specific overlappingpeptide pool. After 48 h of stimulation, supernatant was collected andcytokine concentrations were determined. One point in the graph standsfor the individual cytokine concentration of one mouse, every mousesample was measured in duplicates (group size n=8; mean is included forthe groups).

FIG. 30 : Schematic overview of the S protein organization of theSARS-CoV-2 S protein and novel constructs for the development of aSARS-CoV-2 vaccine.

Based on the wildtype S protein, we have designed two differenttransmembrane-anchored RBD-based vaccine constructs encoding the RBDfragment fused to the T4 fibritin trimerization domain (F) and theautochthonus transmembrane domain (TM). Construct (1) starts with theSARS-CoV-2-S signal peptide (SP; AA 1-19 of the S protein) whereasconstruct (2) starts with the human Ig heavy chain signal peptide(huSec) to ensure Golgi transport to the cell membrane.

FIG. 31 : Anti-S protein IgG response 6, 14 and 21 d after immunizationwith LNP-C12 formulated modRNA coding for transmembrane-anchoredRBD-based vaccine constructs.

BALB/c mice were immunized IM once with 4 μg of LNP-C12-formulatedtransmembrane-anchored RBD-based vaccine constructs (surrogate toBNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animalswere bled and the serum samples were analyzed for total amount ofanti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G(IgG) measured via ELISA. For day 6 (1:50), day 14 (1:300) and day 21(1:900) different serum dilution were included in the graph. One pointin the graph stands for one mouse, every mouse sample was measured induplicates (group size n=8; mean+SEM is included for the groups).

FIG. 32 : Neutralization of SARS-CoV-2 pseudovirus 6, 14 and 21 d afterimmunization with LNP-C12 formulated modRNA coding fortransmembrane-anchored RBD-based vaccine constructs.

BALB/c mice were immunized IM once with 4 μg of LNP-C12-formulatedtransmembrane-anchored RBD-based vaccine constructs (surrogate toBNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animalswere bled and the sera were tested for SARS CoV-2 pseudovirusneutralization. Graphs depict pVN50 serum dilutions (50% reduction ofinfectious events, compared to positive controls without serum). Onepoint in the graphs stands for one mouse. Every mouse sample wasmeasured in duplicate. Group size n=8. Mean+SEM is shown by horizontalbars with whiskers for each group. LLOQ, lower limit of quantification.ULOQ, upper limit of quantification.

FIG. 33 : Immunogenicity of BNT162b1 in Rhesus macaques and comparisonto human convalescent sera.

Rhesus macaques were immunized IM on days 0 and 21 with 30 μg or 100 μgof BNT162b1 or with placebo (0.9% NaCl). Sera were obtained beforeimmunization and 14, 21, 28, and 35 days after immunization; PBMCs wereobtained before and 14 and 42 days after immunization. Sera fromCOVID-19 patients were obtained 20-40 days after the onset of symptomsand after at least 14 days of asymptomatic convalescence. a, Geometricmean concentrations of IgG binding to a recombinant S1 protease fragmentof SARS-CoV-2 S, in Rhesus macaque sera drawn at the indicated timesafter immunization (n=6 per group, all measurement time points of theplacebo group depicted under ‘Control’) and in human convalescent sera(n=62). b, SARS-CoV-2 geometric mean 50% neutralization titers of theRhesus macaque sera (n=6 per group, all measurement time points of theplacebo group depicted under ‘Control’) and human convalescent sera(n=38). P values were determined by a two-tailed one-way ANOVA andDunnett's multiple comparisons test. c, Flow cytometry analysis of CD4⁺T cells producing IFNγ, IL-2, TNF (T_(H)1), IL-21 or IL-4 (T_(H)2)cytokines in the Rhesus macaque PBMCs on day 42. P values weredetermined by a two-tailed Kruskal-Wallis test followed by Dunn'smultiple comparisons test. Each data point corresponds to an individualanimal.

FIG. 34 : Overview of study population

FIG. 35 : Local Reactions Reported within 7 Days of Vaccination all DoseLevels Solicited injection-site (local) reactions were: pain atinjection site (mild=does not interfere with activity;moderate=interferes with activity; severe=prevents daily activity; Grade4=emergency room visit or hospitalization) and redness and swelling(mild=2.5 to 5.0 cm in diameter; moderate=5.5 to 10.0 cm in diameter;severe=>10.0 cm in diameter; Grade 4=necrosis or exfoliative dermatitisfor redness, and necrosis for swelling). Data were collected with theuse of electronic diaries for 14 days after each vaccination.

FIG. 36 : a: Systemic Events Reported within 7 days after Vaccination 1:All Dose Levels; b: Systemic Events Reported within 7 days afterVaccination 2: 10 μg & 30 μg Dose Levels

Solicited systemic events were: nausea/vomiting (mild=no interferencewith activity or 1 to 2 times in 24 hours; moderate=some interferencewith activity or >2 times in 24 hours; severe=prevents daily activity orrequires intravenous hydration; Grade 4=emergency room visit orhospitalization for hypotensive shock), diarrhea (mild, 2 to 3 loosestools in 24 hours; moderate, 4 to 5 loose stools in 24 hours; severe,≥6 loose stools in 24 hours; Grade 4=emergency room visit orhospitalization), headache (mild=no interference with activity;moderate=repeated use of non-narcotic pain reliever >24 hours or someinterference with activity; severe=significant, any use of narcotic painreliever or prevents daily activity; Grade 4=emergency room visit orhospitalization), fatigue/tiredness (mild=no interference with activity;moderate=some interference with activity; severe=significant; preventsdaily activity; Grade 4=emergency room visit or hospitalization), musclepain (pain that is occurring in areas other than the injection site;mild=no interference with activity; moderate=some interference withactivity; severe=significant; prevents daily activity; Grade 4=emergencyroom visit or hospitalization), joint pain (mild=no interference withactivity; moderate=some interference with activity; severe=significant;prevents daily activity; Grade 4=emergency room visit orhospitalization), and fever (mild=100.4° F. to 101.1° F. [38.0° C. to38.4° C.]; moderate=101.2° F. to 102.0° F. [38.5° C. to 38.9° C.];severe=102.1° F. to 104.0° F. [39.0° C. to 40.0° C.]; Grade 4=>104.0° F.[>40.0° C.]).

FIG. 37 : Immunogenicity of BNT162b1—RBD-Binding IgG GMCs and SARS CoV250% Neutralizing Titers after 1 or 2 doses

Subjects in groups of 15 were immunized with the indicated dose levelsof BNT162b1 (n=12) or with placebo (P, n=3) on days 1 (all dose levelsand placebo) and 21 (10 μg and 30 μg dose levels and placebo). Sera wereobtained before immunization (Day 1) and 7, 21, and 28 days after thefirst immunization. Human COVID-19 convalescent sera (HCS) (n=38) wereobtained 20-40 days after the onset of symptoms and after at least 14days of asymptomatic convalescence. a, GMCs of recombinant RBD-bindingIgG. Lower limit of quantitation (LLOQ) 1.15 (dotted line). b, 50%SARS-CoV-2 neutralizing GMTs. Each data point represents a serum sample,and each vertical bar represents a geometric mean with 95% confidenceinterval.

FIG. 38 : BNT162b1 induces strong CD4 and CD8 T cell response in humans

BNT162 induced T cells: INFγ ELISpot ex vivo; T cell responses in 8 of 8tested subjects. Here: subject vaccinated prime/boost with 10 μgBNT162b1; CEF: CMV, EBV, Influenza CD8 T cell epitope mix, CEFT: CMV,EBV, Influenza, Tetanus CD4 T cell epitope mix.

FIG. 39 : BNT162b1-induced IgG concentrations

Subjects were immunised with BNT162b1 on days 1 (all dose levels) and 22(all dose levels except 60 μg) (n=12 per group, from day 22 on n=11 forthe 10 μg and 50 μg cohort). Sera were obtained on day 1 (Pre prime) andon day 8, 22 (pre boost), 29 and 43. Pre-dose responses across all doselevels were combined. Human COVID-19 convalescent sera (HCS, n=38) wereobtained at least 14 days after PCR-confirmed diagnosis and at a timewhen the donors were no longer symptomatic. For RBD-binding IgGconcentrations below the lower limit of quantification (LLOQ=1.15),LLOQ/2 values were plotted. Arrowheads indicate vaccination. Chequeredbars indicate that no boost immunisation was performed. Values abovebars are geometric means with 95% confidence intervals. At the time ofsubmission, day 43 data were pending for five subjects of the 50 μgcohort and all subjects of the 60 μg cohort.

FIG. 40 : BNT162b1-induced virus neutralisation titers

The vaccination schedule and serum sampling are the same as in FIG. 39 .a, SARS-CoV-2 50% neutralisation titers (VNT₅₀) in immunized subjectsand COVID-19 convalescent patients (HCS). For values below the lowerlimit of quantification (LLOQ)=20, LLOQ/2 values were plotted.Arrowheads indicate days of immunisation. Chequered bars indicate thatno boost immunisation was performed. Geometric mean (values above bars)with 95% confidence interval. At the time of submission, day 43 datawere not yet available for five subjects of the 50 μg cohort and allsubjects of the 60 μg cohort, b, Correlation of RBD-binding IgGgeometric mean concentrations (GMC) (as in FIG. 39 ) with VNT₅₀ on day29 (all evaluable subject sera). Nonparametric Spearman correlation. c,Pseudovirus 50% neutralisation titers (pVNT₅₀) across a pseudoviruspanel displaying 17 SARS-CoV-2 spike protein variants including 16 RBDmutants and the dominant spike protein variant D614G (dose level 10, 30and 50 μg, n=1-2 each; day 29). Lower limit of quantification (LLOQ)=40.Geometric mean.

FIG. 41 : Frequency and magnitude of BNT162b1-induced CD4⁺ and CD8⁺T-cell responses

The vaccination schedule is as in FIG. 39 . PBMCs obtained on day 1(Pre) and on day 29 (Post, 7 days after boost) (1 and 50 μg, n=8 each;10 and 30 μg, n=10 each) were enriched for CD4⁺ or CD8⁺ T cell effectorsand separately stimulated over night with an overlapping peptide poolrepresenting the vaccine-encoded RBD for assessment in direct ex vivoIFNγ ELISpot. Common pathogen T-cell epitope pools CEF (CMV, EBV,influenza virus HLA class I epitopes) and CEFT (CMV, EBV, influenzavirus, tetanus toxoid HLA class II epitopes) served to assess generalT-cell reactivity, medium served as negative control. Each dotrepresents the normalized mean spot count from duplicate wells for onestudy subject, after subtraction of the medium-only control. a, Ratiosabove post-vaccination data points are the number of subjects withdetectable CD4⁺ or CD8⁺ T cell response within the total number oftested subjects per dose cohort. b, Exemplary CD4⁺ and CD8⁺ ELISpot of a10-μg cohort subject. c, RBD-specific CD4⁺ and CD8⁺ T cell responses inall prime/boost vaccinated subjects and their baseline CEFT- andCEF-specific T-cell responses. d, Correlation of VNT₅₀ (as in FIG. 40 a) with CD4⁺ T-cell responses (as in FIG. 41 ) of dose cohorts 10 to 50μg (1 and 50 μg, n=8 each; 10 and 30 μg, n=10 each). NonparametricSpearman correlation.

FIG. 42 : Cytokine polarisation of BNT162b1-induced T cells

The vaccination schedule and PBMC sampling are as in FIG. 41 . PBMCs ofvaccinees and COVID-19 recovered donors (HCS n=6; in (c)) werestimulated over night with an overlapping peptide pool representing thevaccine-encoded RBD and analysed by flow cytometry (a-c) and bead-basedimmunoassay (d). a, Exemplary pseudocolor flow cytometry plots ofcytokine-producing CD4⁺ and CD8⁺ T cells of a 10-μg cohort subject. b,RBD-specific CD4⁺ T cells producing the indicated cytokine as fractionof total cytokine-producing RBD-specific CD4⁺ T cells, and c,RBD-specific CD8⁺ (left) or CD4⁺ (right) T cells producing the indicatedcytokine as fraction of total circulating T cells of the same subset.One CD4 non-responder (<0.02% total cytokine producing T cells) and oneCD8 non-responder (<0.01% total cytokine producing T cells) from the30-μg cohort were excluded in (b). Values above data points are the meanfractions across all dose cohorts. d, PBMCs from the 50-μg cohort. Eachdot represents the mean from duplicate wells subtracted by the DMSOcontrol for one study subject. Lower limits of quantification (LLOQ)were 6.3 pg/mL for TNF, 2.5 pg/mL for IL-1S, and 7.6 pg/mL for IL-12p70.Mean (b).

FIG. 43 : Schedule of vaccination and assessment

FIG. 44 : Solicited adverse events

Subjects were immunized with the indicated dose levels of BNT162b1 ondays 1 (all dose levels) and 22 (all dose levels except 60 μg) (n=12 pergroup, n=11 for 10 μg and 50 μg cohort from day 22 on). a, b, Number ofsubjects with local (a) or systemic reactions (b) by day (day 1-9,22-30) and cohort. Grading of adverse events was performed according toFDA recommendations (U.S. Department of Health and Human Services,Administration, F. and D. & Research, C. for B. E. and. Toxicity gradingscale for healthy adult and adolescent volunteers enrolled in preventivevaccine clinical trials. (2007). Available at:www.fda.gov/regulatory-information/search-fda-guidance-documents/toxicity-grading-scale-healthy-adult-and-adolescent-volunteers-enrolled-preventive-vaccine-clinical).

FIG. 45 : Pharmacodynamic markers

Subjects were immunised with the indicated dose levels of BNT162b1 ondays 1 (all dose levels) and 22 (all dose levels except 60 μg). a,Kinetics of C-reactive protein (CRP) level and b, Kinetics of lymphocytecounts. Dotted lines indicate upper and lower limit of reference range.For values below the lower limit of quantification (LLOQ=0.3), LLOQ/2values were plotted (a).

FIG. 46 : Correlation of antibody and T-cell responses

Subjects were immunised with the indicated dose levels of BNT162b1 ondays 1 (all dose levels) and 22 (all dose levels except 60 μg). a,Correlation of RBD-specific IgG responses (from FIG. 39 a ) with CD4⁺T-cell responses on day 29 (1 and 50 μg, n=8 each; 10 and 30 μg, n=10each). Nonparametric Spearman correlation. b, Correlation of CD4⁺ withCD8⁺ T-cell responses (as in FIG. 41 ) from day 29 of dose cohorts 10 to50 μg (1 and 50 μg, n=8 each; 10 and 30 μg, n=10 each). ParametricPearson correlation. c, Correlation of RBD-specific IgG responses (fromFIG. 39 a ) with CD8⁺ T-cell responses on day 29 (1 and 50 μg, n=8 each;10 and 30 μg, n=10 each). Nonparametric Spearman correlation.

FIG. 47 : Gating strategy for flow cytometry analysis of data shown inFIG. 42

Flow cytometry gating strategy for identification of IFNγ, IL-2 and IL-4secreting T cells in study subject PBMC samples. a, CD4⁺ and CD8⁺ Tcells were gated within single, viable lymphocytes. b, c, Gating ofIFNγ, IL-2 and IL-4 in CD4⁺ T cells (b), and IFNγ and IL-2 in CD8⁺ Tcells (c).

FIG. 48 : BNT162b1 18-55 years of age: Local Reactions After Each Dose

FIG. 49 : BNT162b1 18-55 years of age: Systemic Events After Each Dose

FIG. 50 : BNT162b1 65-85 years of age: RBD-Binding IgG GMCs

FIG. 51 : BNT162b1 65-85 years of age: 50% SARS-CoV-2 Neutralizing GMTs

FIG. 52 : BNT162b2 18-55 years of age: Local Reactions After Each Dose

FIG. 53 : BNT162b2 18-55 years of age: Systemic Events After Each Dose

FIG. 54 : BNT162b2 65-85 years of age: Local Reactions After Each Dose

FIG. 55 : BNT162b2 65-85 years of age: Systemic Events After Each Dose

FIG. 56 : BNT162b2 18-55 years of age: S1-Binding IgG GMCs

FIG. 57 : BNT162b2 18-55 years of age: 50% SARS-CoV-2 Neutralizing GMTs

FIG. 58 : BNT162b2 65-85 years of age: S1-Binding IgG GMCs

FIG. 59 : BNT162b2 65-85 years of age: 50% SARS-CoV-2 Neutralizing GMTs

FIG. 60 : BNT162b2-elicited T cell responses in mice

Splenocytes of BALB/c mice immunized IM with BNT162b2 or buffer were exvivo restimulated with full-length S peptide mix or negative controls(irrelevant peptide in a, right); no peptide in (a, left) and in (c)).P-values were determined by a two-tailed paired t-test. (a) IFNγ ELISpotof splenocytes collected 12 days after immunization of mice (n=8 pergroup) with 5 μg BNT162b2 (left). IFNγ ELISpot of isolated splenic CD4+T cells or CD8+ T cells 28 days after immunization of mice (n=8 mice pergroup) with 1 μg BNT162b2 (middle and right). (b) CD8+ T-cell specificcytokine release by splenocytes of mice (n=8 per group) immunized with 5μg BNT162b2 or buffer (control), determined by flow cytometry. S-peptidespecific responses are corrected for background (no peptide). (c)Cytokine production by splenocytes obtained 28 days after immunizationof mice (n=8 per group, n=7 for IL-4, IL-5, and IL-13, as one outlierwas removed via routs test [Q=1%] for the S peptide stimulated samples)with 1 μg BNT162b2, determined by bead-based multiplex analysis.

FIG. 61 : IFNγ ELiSpot data for 5 subjects vaccinated with 10 μgBNT162b2

Background-subtracted spot counts from duplicates prior to vaccination(Pre) and on day 29 (Post-7 days post boost) per 10⁶ cells. T cellresponse analysis was performed in a GCLP-compliant manner using avalidated ex-vivo IFNγ ELISpot assay. All tests were performed induplicate and included negative and positive controls (medium only andanti-CD3). In addition, peptide epitopes derived from cytomegalovirus(CMV), Epstein Barr virus (EBV), and influenza virus were used aspositive controls. CD4- or CD8-depleted PBMCs were stimulated for 16-20h in pre-coated ELISpot plates (Mabtech) with overlapping peptidescovering the N-terminal portion and C-terminal portion of the spikeglycoprotein. For analysis of ex vivo T-cell responses, bound IFNγ wasvisualized by an alkaline phosphatase-conjugated secondary antibody.Plates were scanned using a Robot ELISPOT Reader and analysed byImmunoCapture V6.3 or AID ELISPOT 7.0 software. Spot counts weresummarized as mean values for each duplicate. T cell counts werecalculated as the sum of spot counts detected after stimulation with Spool 1 and S pool 2. T-cell responses stimulated by peptides werecompared to effectors incubated with medium only as negative controlusing an ELISpot data analysis Tool (EDA), based on two statisticaltests (distribution free resampling) according to Moodie et al. (MoodieZ. et al., J Immunol Methods 315, 2006, 121-32; Moodie Z. et al., CancerImmunol Immunother 59, 2010, 1489-501) thus providing sensitivity whilemaintaining control over false positive rate. No significant changeswere observed between the pre- and day 29 T cell responses against thepositive control peptides from CMV, EBV, and influenza virus (notshown).

FIG. 62 : Example of CD4⁺ and CD8⁺ IFNγ ELiSpot data

IFNγ ELISpot was performed as in FIG. 61 using PBMCs obtained from asubject prior to immunization and on day 29 after dose 1 of 10 μgBNT162b2 (7 days post dose 2). HLA class I and class II peptide poolsCEF (cytomegalovirus [CMV], Epstein Barr virus [EBV] (7 days post dose2), and influenza virus, HLA class I epitope mix) and CEFT (CMV, EBV,influenza virus, and tetanus toxoid HLA class II cell epitope mix) wereused as benchmarking controls to assess CD8+ and CD4+ T cell reactivity.

FIG. 63 : Comparison of BNT162b2-elicited and benchmark INFγ ELISpotresponses

IFNγ spot counts from day 29 (7 day post dose 2) PBMC samples obtainedfrom 5 subjects who were immunized with 10 μg of BNT162b2 on days 1 and22. CEF (CMV, EBV, and influenza virus HLA class I epitope mix), andCEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class II cellepitope mix) were used as benchmarking controls to assess CD8⁺ and CD4⁺T cell reactivity. Horizontal lines indicate median values.

FIG. 64 : Design and characterisation of the immunogen

a, Structure of BNT162b1. Linear diagram of RNA (left), and cartoon ofLNP (right). UTR, untranslated region; SP, signal peptide. b,Representative 2D class averages from electron microscopy of negativelystained RBD-foldon trimers. Box edge: 37 nm. c, Density map of theACE2/B⁰AT1/RBD-foldon trimer complex at 3.24 Å after focused refinementof the ACE2 extracellular domain bound to an RBD monomer. Surfacecolor-coding by subunit. A ribbon model refined to the density shows theRBD-ACE2 binding interface, with residues potentially mediating polarinteractions labeled.

FIG. 65 : Mouse immunogenicity

a-c, BALB/c mice (n=8 per group) were immunised intramuscularly (IM)with 0.2, 1 or 5 μg of BNT162b1 or buffer. Geometric mean of eachgroup±95% CI, P-values compare day 28 to non-immunised (0 μg; n=8)baseline sera (multiple comparison of mixed-effect analysis usingDunnett's multiple comparisons test) (a, c). a, RBD-binding IgGresponses in sera obtained 7, 14, 21 and 28 days after immunisation,determined by ELISA. For day 0, a pre-screening of randomised animalswas performed (n=4). b, Representative surface plasmon resonancesensorgram of the binding kinetics of His-tagged RBD to immobilisedmouse IgG from serum 28 days after immunisation with 5 μg BNT162b1(n=8). Actual binding (green) and the best fit of the data to a 1:1binding model (black). c, VSV-SARS-CoV-2 pseudovirus 50% serumneutralising titers (pVNT₅₀). d-f, Splenocytes of BALB/c mice immunisedIM with BNT162b1 or buffer (control) were ex vivo re-stimulated withfull-length S peptide mix or negative controls (no peptide in (d, left)and in (e, f); irrelevant peptide in (d, right)). P-values weredetermined by a two-tailed paired t-test. d, IFNγ ELISpot of splenocytescollected 12 days after immunisation of mice (n=8 per group) with 5 μgBNT162b1 (left). IFNγ ELISpot of isolated splenic CD4⁺ T cells (n=7, oneoutlier removed by Grubbs test, α=0.05) or CD8⁺ T cells (n=8) 28 daysafter immunisation with 1 μg BNT162b1 (middle and right). e, T-cellspecific cytokine release by splenocytes of mice (n=8 per group)immunised with 5 μg BNT162b1, determined by flow cytometry. S-peptidespecific responses are corrected for background (no peptide). f,Cytokine production by splenocytes obtained 28 days after immunisationof mice (n=8 per group) with 0.2 μg BNT162b1, determined by bead-basedmultiplex analysis.

FIG. 66 : Immunogenicity of BNT162b1 in Rhesus macaques and comparisonto human convalescent sera

a, b, Male Rhesus macaques 2-4 years of age (n=6 per group) wereimmunised IM on Days 0 and 21 with 30 μg or 100 μg of BNT162b1 or withbuffer, and serum was obtained before and 14, 21, 28, 35 and 42 daysafter immunisation. Human convalescent sera (HCS) were obtained fromSARS-CoV-2-infected patients at least 14 days after PCR-confirmeddiagnosis and at a time when acute COVID-19 symptoms had resolved(n=38). Values above bars give the geometric means. a, Geometric meanconcentrations (GMCs) of IgG binding a recombinant SARS-CoV-2 RBD.Dashed line indicates geometric mean of sera from all time points forthe placebo group (1.72 U/mL). Group IgG titers for every time pointwere analysed for statistical significance against HCS samples usingone-way ANOVA with Dunnett's multiple comparison correction, andstatistical significance was confirmed in the 30 μg dose-level group(Day 28, p<0.0001; Day 35, p=0.0016), and in the 100 μg dose-level group(Day 28, 35 and 42, all p<0.0001). b, SARS-CoV-2 50% neutralisationtiters (VNT₅₀). Dashed line indicates geometric mean of sera from alltime points for the placebo group (10.31 U/mL). Group VNT₅₀ for everytime point were analysed for statistical significance against HCSsamples using one-way ANOVA with Dunnett's multiple comparisoncorrection, and statistical significance was confirmed in the 30 μgdose-level group (Day 28, p<0.0001), and in the 100 μg dose-level group(Day 28 and 35, both p<0.0001; Day 42, p=0.007).

FIG. 67 : Viral RNA in non-immunised and immunised Rhesus macaques afterSARS-CoV-2 challenge

Rhesus macaques (n=6 per group) were immunised on Days 0 and 21 with 100μg BNT162b1 or buffer (Control) as described in FIG. 66 . Forty-one to48 days after the second immunisation, the animals were challenged with1×10⁶ total pfu of SARS-CoV-2 split equally between the IN and ITroutes. Three non-immunised age-matched male Rhesus macaques werechallenged with cell culture medium (Sentinel). Viral RNA levels weredetected by RT-qPCR. Ratios above data points are the number of viralRNA positive animals within all animals per group. a, Viral RNA inbronchoalveolar lavage (BAL) fluid obtained before, and on Days 3 and 6after challenge. At day 6, the viral load between the control andBNT162b1-immunized animals was statistically significant (p=0.0131). b,Viral RNA in nasal swabs obtained before challenge and on day 1, 3, and6 after challenge. At day 3, the viral load between the control andBNT162b1-immunized animals was statistically significant (p=0.0229).Dotted lines indicate the lower limits of detection (LLOD). Negativespecimens were set to 2 the LLOD. P-values were determined bycategorical analysis for binomial response (undetectable viral loadafter challenge as success, measurable viral load after challenge asfailure).

FIG. 68 : BNT162b1 and b2 V8 immunization reduces viral RNA in Rhesusmacaques after challenge with SARS-CoV-2; b2 shows earlier clearance innose

FIG. 69 : Exemplary pandemic supply product packaging overview

FIG. 70 : Exemplary vaccine storage & handling at the point ofvaccination

FIG. 71 : Exemplary multi-dose preparation

FIG. 72 . Geometric Mean Titers and 95% CI: SARS-CoV-2 NeutralizationAssay-NT50-Phase 1, 2 Doses, 21 Days Apart-18-55 Years ofAge-BNT162b1-Evaluable Immunogenicity Population

FIG. 73 . Geometric Mean Titers and 95% CI: SARS-CoV-2 NeutralizationAssay-NT50-Phase 1, 2 Doses, 21 Days Apart-65-85 Years ofAge-BNT162b1-Evaluable Immunogenicity Population

FIG. 74 . Geometric Mean Titers and 95% CI: SARS-CoV-2 NeutralizationAssay-NT50-Phase 1, 2 Doses, 21 Days Apart-18-55 Years ofAge-BNT162b2-Evaluable Immunogenicity Population

FIG. 75 . Geometric Mean Titers and 95% CI: SARS-CoV-2 NeutralizationAssay-NT50-Phase 1, 2 Doses, 21 Days Apart-65-85 Years ofAge-BNT162b2-Evaluable Immunogenicity Population

FIG. 76 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2RBD-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-18-55 Yearsof Age-BNT162b1-Evaluable Immunogenicity Population

FIG. 77 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2RBD-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-65-85 Yearsof Age, BNT162b1-Evaluable Immunogenicity Population

FIG. 78 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2S1-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-18-55 Yearsof Age-BNT162b1-Evaluable Immunogenicity Population

FIG. 79 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2S1-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-65-85 Yearsof Age-BNT162b1-Evaluable Immunogenicity Population

FIG. 80 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2S1-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-18-55 Yearsof Age-BNT162b2-Evaluable Immunogenicity Population

FIG. 81 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2S1-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-65-85 Yearsof Age-BNT162b2-Evaluable Immunogenicity Population

FIG. 82 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2RBD-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-18-55 Yearsof Age-BNT162b2-Evaluable Immunogenicity Population

FIG. 83 . Geometric Mean Concentrations and 95% CI: SARS-CoV-2RBD-binding IgG Level Assay-Phase 1, 2 Doses, 21 Days Apart-65-85 Yearsof Age-BNT162b2-Evaluable Immunogenicity Population

FIG. 84 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-18-55Years of Age-BNT162b1-Safety Population

FIG. 85 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-65-85Years of Age-BNT162b1-Safety Population

FIG. 86 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-18-55Years of Age-BNT162b2-Safety Population

FIG. 87 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-65-85Years of Age-BNT162b2-Safety Population

FIG. 88 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-18-55Years of Age-BNT162b1-Safety Population

FIG. 89 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-65-85Years of Age-BNT162b1-Safety Population

FIG. 90 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-18-55Years of Age-BNT162b2-Safety Population

FIG. 91 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose-Phase 1, 2 Doses, 21 Days Apart-65-85Years of Age-BNT162b2-Safety Population

FIG. 92 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose, Age Group 18 55 Years-Phase 2-SafetyPopulation

FIG. 93 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose, Age Group 56 85 Years-Phase 2-SafetyPopulation

FIG. 94 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose, Age Group 18 55 Years-Phase 2-SafetyPopulation

FIG. 95 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose, Age Group 56 85 Years-Phase 2-SafetyPopulation

FIG. 96 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose, Age Group 18 55 Years-^(˜)6000 Subjectsfor Phase 2/3-Safety Population

FIG. 97 . Subjects Reporting Local Reactions, by Maximum Severity,Within 7 Days After Each Dose, Age Group 56 85 Years-^(˜)6000 Subjectsfor Phase 2/3-Safety Population

FIG. 98 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose, Age Group 18-55 Years-^(˜)6000 Subjectsfor Phase 2/3-Safety Population

FIG. 99 . Subjects Reporting Systemic Events, by Maximum Severity,Within 7 Days After Each Dose, Age Group 56-85 Years-^(˜)6000 Subjectsfor Phase 2/3-Safety Population

FIG. 100 . Cumulative Incidence Curves for the First COVID-19 OccurrenceAfter Dose 1-Dose 1 All-Available Efficacy Population

FIG. 101 . BNT162b2—Exemplary functional 50% SARS-CoV-2 neutralisingantibody titers (VN₅₀).

Younger adults (aged 18 to 55 years) and older adults (aged 56 to 85years) were immunized with BNT162b2 on day 1 and day 22 (n=12 pergroup). Sera were obtained from younger adults on day 1 (baseline) andon day 8, 22 (pre boost), 29, 43, 50 and 85. Sera were obtained fromolder adults on day 1 (baseline) and on day 8, 22, and 29. HumanCOVID-19 convalescent sera (HSC, n=38) were obtained at least 14 daysafter a confirmed diagnosis and at a time when the donors were no longersymptomatic. SARS-CoV-2 50% neutralization titers (VN₅₀ titers) with 95%confidence intervals are shown for younger adults immunized with 1, 3,10, 20, or 30 μg BNT162b2, and older adults immunized with 20 μgBNT162b2. Values smaller than the limit of detection (LOD) are plottedas 0.5*LOD. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2(Day 22). The dotted horizontal line represents the LOD. VN₅₀=50%SARS-CoV-2 neutralizing antibody titers; HCS=human COVID-19 convalescentserum.

FIG. 102 . BNT162b1—Exemplary fold increase from baseline in functional50% SARS-CoV-2 neutralizing antibody titers (VN₅₀).

The vaccination schedule and serum sampling are the same as in FIG. 39(n=12 per group). Geometric means fold increase (GMFI) from baseline inVN₅₀ titer with 95% confidence intervals are shown for youngerparticipants (aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 μgBNT162b1. Arrowheads indicate baseline (pre-dose 1, Day 1) and dose 2(Day 22). Dose 2 was not performed in the 60 μg dose group. The dottedhorizontal line represents the threshold for seroconversion (foldincrease≥4). VN₅₀=50% SARS-CoV-2 neutralizing antibody titers.

FIG. 103 . BNT162b2—Exemplary fold increase from baseline in functional50% SARS-CoV-2 neutralizing antibody titers (VN₅₀).

The vaccination schedule and serum sampling are the same as in FIG. 101. Geometric means fold increase (GMFI) from baseline in VN₅₀ titer with95% confidence intervals are shown for (A) younger participants (aged 18to 55 yrs) immunized with 1, 3, 10, 20, or 30 μg BNT162b2, and (B) olderparticipants (aged 56 to 85 yrs) immunized with 20 μg BNT162b2.Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22).The dotted horizontal line represents the threshold for seroconversion(fold increase≥4). VN₅₀=50% SARS-CoV-2 neutralizing antibody titers.

FIG. 104 . Exemplary frequencies of participants with SARS-CoV-2 GMTseroconversion after immunization with BNT162b1.

The vaccination schedule and serum sampling are the same as in FIG. 39(n=12 per group). Seroconversion with regard to 50% SARS-CoV-2neutralizing antibody titers (VN₅₀) is shown for younger participants(aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 μg BNT162b1.Seroconversion is defined as a minimum of a 4-fold increase offunctional antibody response as compared to baseline. Arrowheadsindicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). Dose 2 wasnot performed in the 60 μg dose group. GMT=geometric mean titer.

FIG. 105 . Exemplary frequencies of participants with SARS-CoV-2 GMTseroconversion after immunization with BNT162b2.

The vaccination schedule and serum sampling are the same as in FIG. 101. Seroconversion with regard to 50% SARS-CoV-2 neutralizing antibodytiters (VN₅₀) is shown for (A) younger participants (aged 18 to 55 yrs)dosed with 1, 3, 10, 20, or 30 μg BNT162b2, and (B) older participants(aged 56 to 85 yrs) dosed with 20 μg BNT162b2. Seroconversion is definedas a minimum of 4-fold increase of functional antibody response ascompared to baseline. Arrowheads indicate baseline (pre-Dose 1, Day 1)and Dose 2 (Day 22). GMT=geometric mean titer.

FIG. 106 . Exemplary fold increase from baseline in S1-binding antibodyconcentrations after immunization with BNT162b1.

The vaccination schedule and serum sampling are the same as in FIG. 39(n=12 per group). Geometric means fold increase (GMFI) from baseline inS1-binding antibody concentrations with 95% confidence intervals areshown for younger participants (aged 18 to 55 yrs) immunized with 1, 10,30, 50, or 60 μg BNT162b1. Arrowheads indicate baseline (pre-Dose 1,Day 1) and Dose 2 (Day 22). Dose 2 was not performed in the 60 μg dosegroup. The dotted horizontal line represents the threshold forseroconversion (fold increase≥4).

FIG. 107 . Exemplary fold increase from baseline in S1-binding antibodyconcentration after immunization with BNT162b2.

The vaccination schedule and serum sampling are the same as in FIG. 101. Geometric means fold increase (GMFI) from baseline in S1-bindingantibody concentrations with 95% confidence intervals are shown for (A)younger participants (aged 18 to 55 yrs) immunized with 1, 3, 10, 20, or30 μg BNT162b2, and (B) older participants (aged 56 to 85 yrs) immunizedwith 20 μg BNT162b2. Arrowheads indicate baseline (pre-Dose 1, Day 1)and Dose 2 (Day 22). The dotted horizontal line represents the thresholdfor seroconversion (fold increase≥4).

FIG. 108 . Exemplary frequencies of participants with S1-binding IgG GMCseroconversion after immunization with BNT162b1.

The vaccination schedule and serum sampling are the same as in FIG. 39(n=12 per group). Seroconversion with regard to S1-binding antibody GMCis shown for younger participants (aged 18 to 55 yrs) immunized with 1,10, 30, 50, or 60 μg BNT162b1. Seroconversion is defined as at least a4-fold increase of S1-binding IgG GMC response as compared to baseline.Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22).Dose 2 was not performed in the 60 μg dose group. GMC=geometric meanconcentration.

FIG. 109 . Exemplary frequencies of participants with S1-binding IgG GMCseroconversion after immunization with BNT162b2.

The vaccination schedule and serum sampling are the same as in FIG. 101. Seroconversion with regard to S1-binding antibody GMC is shown for (A)younger participants (aged 18 to 55 yrs) immunized with 1, 3, 10, 20, or30 μg BNT162b2, and (B) older participants (aged 56 to 85 yrs) dosedwith 20 μg BNT162b2. Seroconversion is defined as at least a 4-foldincrease of S1-binding IgG GMC response as compared to baseline.Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22).GMC=geometric mean concentration

FIG. 110 . Exemplary results of cytokine production produced fromS-specific CD4⁺ T cells from younger participants immunized withBNT162b2.

Peripheral blood mononuclear cell (PBMC) cell fractions isolated fromblood of participants treated with varying doses of BNT162b2 werecollected at baseline (pre-Dose one) and 29 days (±3 days) after Doseone and analyzed. Participants included younger participants (age 18-55years) dosed at 1 μg (n=8), 3 μg (n=9), 10 μg (n=10), 20 μg (n=9), or 30μg (n=10). Bar charts show arithmetic means with 95% confidenceinterval. Cytokine production was calculated by summing up the fractionsof all CD4⁺ T cells positive for either IFNγ, IL-2, or IL-4, settingthis sum to 100% and calculating the fraction of each specificcytokine-producing subset thereof. Two participants from the 1 μgcohort, 1 participant from the 3 μg cohort, and 1 participant from the10 μg cohort were excluded from this analysis (frequency of totalcytokine-producing CD4⁺ T cells<0.03%). IFN=interferon; IL=interleukin;younger participants=participants aged 18 to 55 yrs; Sprotein=SARS-CoV-2 spike protein.

FIG. 111 . Exemplary results of cytokine production produced fromS-specific CD4⁺ T cells from older participants immunized with BNT162b2.

Peripheral blood mononuclear cell (PBMC) cell fractions isolated fromblood of participants treated with varying doses of BNT162b2 werecollected at baseline (pre-Dose one) and 29 days (±3 days) after Doseone and analyzed. Participants included older participants (age 56-85years) dosed at 10 μg (n=11), 20 μg (n=8), or 30 μg (n=9). Bar chartsshow arithmetic means with 95% CI. Cytokine production was calculated bysumming up the fractions of all CD4⁺ T cells positive for either IFNγ,IL-2, or IL-4, setting this sum to 100%, and calculating the fraction ofeach specific cytokine-producing subset thereof. Six participants fromthe 10 μg cohort and 1 participant from the 20 μg cohort were excludedfrom this analysis (frequency of total cytokine-producing CD4⁺ Tcells<0.03%). IFN=interferon; IL=interleukin; olderparticipants=participants aged 56 to 85 yrs; S protein=SARS-CoV-2 spikeprotein.

FIG. 112 . Incidence and magnitude of BNT162b2-induced T-cell responses.

PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) (dosecohorts 1, 10 and 20 μg, n=9 each; 30 μg, n=10) were enriched for CD4⁺or CD8⁺ T cell effectors and separately stimulated over night with threeoverlapping peptide pools representing different portions of thewild-type sequence of SARS-CoV-2 S (N-terminal pools S pool 1 and RBD,and the C-terminal S pool 2), for assessment in direct ex vivo IFNγELISpot. Common pathogen T-cell epitope pools CEF (immune dominant HLAclass I epitopes of CMV, EBV, influenza virus) and CEFT (immune dominantHLA class II epitopes CMV, EBV, influenza virus, tetanus toxoid) wereused as controls. Cell culture medium served as negative control. Eachdot represents the normalised mean spot count from duplicate wells forone study participant, after subtraction of the medium-only control (a,c). a, Antigen-specific CD4⁺ and CD8⁺ T-cell responses for each dosecohort. The number of participants with a detectable T-cell response onday 29 over the total number of tested participants per dose cohort isprovided. Spot count data from two participants from the 20 μg dosecohort could not be normalised and are not plotted. b, Example of CD4⁺and CD8⁺ ELISpot for a 30 μg dose cohort participant. c, S-specificT-cell responses in all participants who recognised either S peptidepool and their baseline CEFT- and CEF-specific T-cell responses.Horizontal bars indicate median values.

FIG. 113 . BNT162b2-induced S-specific CD8⁺ and CD4⁺ T cells.

CD4⁺ or CD8⁺ T cell effector-enriched fractions of immunisedparticipants derived from PBMCs obtained on day 1 (pre-prime) and day 29(7 days post-boost) (1, 10 and 20 μg dose cohorts, n=9 each; 30 μg dosecohort, n=10) were stimulated overnight with two overlapping peptidepools covering the wild-type SARS-CoV-2 S (S pool 1 and S pool 2) forassessment in direct ex vivo IFNγ ELISpot (a-c). Each dot represents thenormalised mean spot count from duplicate wells for one studyparticipant, after subtraction of the medium-only control. T-cellresponses against S pool 1 and S pool 2 per participant were combined.Spot count data from two participants from the 20 μg dose cohort couldnot be normalised and are not plotted. PBMCs from vaccinatedparticipants on day 29 (7 days post-boost) (dose cohorts 1 μg, n=7; 10and 30 μg, n=10; 20 μg, n=9) were stimulated as described above andanalysed by flow cytometry (d,e). a, S-specific CD4⁺ and CD8⁺ T-cellresponses for each dose cohort. Number of participants with detectableT-cell response on day 29 over the total number of tested participantsper dose cohort is provided. b, Mapping of vaccine-induced responses ofparticipants with evaluable baseline data (n=34 for CD4⁺ and n=37 forCD8⁺ T cell responses) to different portions of S. De novo induced oramplified responses are classified as BNT162b2-induced response; noresponses or pre-existing responses that were not amplified by thevaccinations are classified as no vaccine response (none). c, Responsestrength to S pool 1 in individuals with or without a pre-existingresponse to S pool 2. Data from the 1 μg dose cohort are excluded, as nobaseline response to S pool 2 was present in this dose cohort.Horizontal bars represent median of each group. d, Examples ofpseudocolor flow cytometry plots of cytokine-producing CD4⁺ and CD8⁺ Tcells from a participant prime/boost vaccinated with 30 μg BNT162b2. e,Frequency of vaccine-induced, S-specific IFNγ⁺ CD4⁺ T cells vs. IL4⁺CD4⁺ T cells. ICS stimulation was performed using a peptide mixture of Spool 1 and S pool 2. Each data point represents one study participant (1μg dose cohort, n=8; 20 μg dose cohort, n=8; 10 and 30 μg, n=10 each).One participant from the 20 μg dose cohort with a strong pre-existingCD4⁺ T cell response to S pool 2 was excluded. f, Antigen-specific CD8⁺T cell frequencies determined by pMHC class I multimer staining (%multimer⁺ of CD8⁺), ICS and ELISpot (% IFNγ⁺ of CD8⁺) for the threeparticipants analysed in FIG. 116 . Signals for S pool 1 and S pool 2were merged.

FIG. 114 . Correlation of antibody and T-cell responses.

Data are plotted for all prime/boost vaccinated participants (dosecohorts 1, 10, 20 and 30 μg) from day 29, with data points forparticipants with no detectable T cell response (open circles; b,c)excluded from correlation analysis. a, Correlation of S1-specific IgGresponses with S-specific CD4⁺ T-cell responses. b, Correlation ofS-specific CD4⁺ with CD8⁺ T-cell responses. c, Correlation ofS1-specific IgG responses with S-specific CD8⁺ T-cell responses.

FIG. 115 . Cytokine polarisation of BNT162b2-induced T cells.

PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) (dosecohorts 1 μg, n=8; 10 and 30 μg, n=10 each; 20 μg, n=9) and COVID-19recovered donors (HCS, n=18; c,d) were stimulated over night with threeoverlapping peptide pools representing different portions of thewild-type sequence of SARS-CoV-2 S (N-terminal pools S pool 1 [aa 1-643]and RBD [aa1-16 fused to aa 327-528 of S], and the C-terminal S pool 2[aa 633-1273]), and analysed by flow cytometry. a, Example ofpseudocolor flow cytometry plots of cytokine-producing CD4⁺ and CD8⁺ Tcells from a 30 μg dose cohort participant in response to S pool 1. b,S-specific CD4⁺ T cells producing the indicated cytokine as a fractionof total cytokine-producing S-specific CD4⁺ T cells in response to Spool 1 and S pool 2. CD4 non-responders (<0.03% total cytokine producingT cells: 1 μg, n=2 [S pool 1] and n=1 [S pool 2]; 10 μg, n=1) wereexcluded. Arithmetic mean with 95% confidence interval. c, S-specificCD4⁺ (S pool 1, S pool 2 and RBD) and d, CD8⁺ T cells (S pool 1, S pool2 and RBD) producing the indicated cytokine as a fraction of totalcirculating T cells of the same subset. Values above data pointsindicate mean fractions per dose cohort. Participant PBMCs were testedas single instance (b-d).

FIG. 116 . Characterization of BNT162b2-induced T cells on the singleepitope level.

PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) ofthree vaccinated participants (dose cohorts 10 μg, n=1; 30 μg, n=2) werestained with individual pMHC class I multimer cocktails and analysed forT cell epitope specificity (a) and phenotype (b; example fromparticipant 3; YLQPRTFLL (SEQ ID NO: 40))) by flow cytometry. Peptidesequences above dot plots indicate pMHC class I multimer epitopespecificity, numbers above dot plots indicate the amino acidscorresponding to the epitope within S. c, Localization of identified MHCclass I-restricted epitopes within S.

FIG. 117 . ELISA screening analysis of exemplary cohort sera to detectantibody responses directed against the recombinant SARS-CoV-2 spikeprotein S1 domain.

ELISA was performed using serum samples collected on day 10 after twoimmunisations (prime/boost on days 1 and 8) with BNT162c1, or on day 17after three administrations (prime/boost on days 1/8/15) of BNT162a1,BNT162b1, or BNT162b2 to analyse elicited antibody responses. The serumsamples were tested against the S1 protein. Group mean ΔOD values ofn=20 mice/group are shown by dots across serum dilutions ranging from1:100 to 1:24,300.

FIG. 118 . ELISA screening analysis of exemplary cohort sera to detectantibody responses directed against the recombinant SARS-CoV-2 spikeprotein RBD domain.

ELISA was performed using serum samples collected on day 10 after twoimmunisations (prime/boost on days 1 and 8) with BNT162c1, or on day 17after three administrations (prime/boost on days 1/8/15) of BNT162a1,BNT162b1, or BNT162b2 to analyse elicited antibody responses. The serumsamples were tested against the RBD domain. Group mean AOD values ofn=20 mice/group are shown by dots across serum dilutions ranging from1:100 to 1:24,300.

FIG. 119 . Pseudovirus neturalisation activity of exemplary cohort seraplotted as pVN₅₀ titre.

Serum samples were collected on day 10 (BNT162c1, saRNA) or day 17 (allother cohorts) after first immunisation of the animals and titres ofvirus-neutralising antibodies were determined by pseudovirus-basedneutralisation test (pVNT). Individual VNT titres resulting in 50%pseudovirus neutralisation (pVN₅₀) are shown by dots; group mean valuesare indicated by horizontal bars (±SEM, standard error of the mean).

FIG. 120 . The virus-neutralising antibodies and specific bindingantibody responses to RBD and S1 in participants.

RBD=receptor binding domain. GMT=geometric mean titer. Serum sampleswere obtained before vaccination (day 1) and day 8, 22, 29, and 43 afterthe prime vaccination in younger adult group, and they were obtainedbefore vaccination (day 1) and day 22, 29, and 43 days after the primevaccination in older adult group. A panel of human COVID-19 convalescentserum (n=24) were obtained at least 14 days after PCR-confirmeddiagnosis in COVID-19 patients. (A) GMTs of SARS-CoV-2 neutralizingantibodies. (B) GMTs of binding antibodies to RBD measured by ELISA. (C)GMTs of ELISA antibodies to S1. Each point represents a serum sample,and each vertical bar represents a geometric mean with 95% CI.

FIG. 121 . T-cell response in participants before and after vaccinationmeasured by IFN-γ ELISpot.

IFN=interferon. PBMC=peripheral blood mononuclear cells. The S1 peptidepool covers the N-terminal half of SARS-CoV-2 spike, including RBD. S2peptide pool covers the C-terminal of SARS-CoV-2 spike, not includingRBD. CEF peptide pool consists of 32 MHC class I restricted viralpeptides from human cytomegalovirus, Epstein-Barr virus and influenzavirus. Panel A shows the number of specific T cell with secretion ofIFN-γ at day 1, 29, and 43 in the younger participants aged 18-55 years.Panel B shows the number of specific T cell with secretion of IFN-γ atday 1, 29, and 43 in the older participants aged 65-85 years.

FIG. 122 . 50% pseudovirus neutralization titers of 16 sera fromBNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudovirus bearingthe Wuhan or lineage B.1.1.7 spike protein. N=8 representative sera eachfrom younger adults (aged 18 to 55 yrs; indicated by triangles) andolder adults (aged 56 to 85 yrs; indicated by circles) drawn at day 43(21 days after dose 2) were tested.

FIG. 123 . Schematic illustration of the production of VSV pseudovirusesbearing SARS-CoV-2 S protein. (1) Transfection of SARS-CoV-2-Sexpression plasmid into HEK293/T17 cells. (2) Infection of SARS-CoV-2 Sexpressing cells with VSV-G complemented input virus lacking the VSV-Gin its genome (VSVΔG) and encoding for reporter genes. (3)Neutralization of residual VSV-G complemented input virus by addition ofanti-VSV-G antibody yields SARS-CoV-2 S pseudotyped VSVΔG as a surrogatefor live SARS-CoV-2.

FIG. 124 . Titration of SARS-CoV-2 Wuhan reference strain and lineageB.1.1.7 spike-pseudotyped VSV on Vero 76 cells using GFP-infected cellsas read-out.

FIG. 125 . Scheme of the BNT162b2 vaccination and serum sampling.

FIG. 126 . Plot of the ratio of pVNT₅₀ between SARS-CoV-2 lineageB.1.1.7 and Wuhan reference strain spike-pseudotyped VSV. Trianglesrepresent sera from younger adults (aged 18 to 55 yrs), and circlesrepresent sera from older adults (aged 56 to 85 yrs). The sea were drawnon day 43 (21 days after dose 2).

FIG. 127 . 50% pseudovirus neutralization titers (pVNT50) of 12 serafrom BNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudovirusbearing the Wuhan Hu-1 reference, lineage B.1.1.298 or lineage B.1.351spike protein.

N=12 sera from younger adults immunized with 30 μg BNT162b2 drawn ateither day 29 or day 43 (7 or 21 days after dose 2) were tested.Geometric mean titers are indicated. Statistical significance of thedifference between the neutralization of the Wuhan Hu-1 referencepseudovirus and either the lineage B.1.1.298 or the lineage B.1.351pseudovirus was calculated by a Wilcoxon matched-pairs signed rank test.Two-tailed p-values are reported. ns, not significant; ***, P<0.001;LLOQ, lower limit of quantification.

FIG. 128 . 50% plaque reduction neutralization titers of 20 sera fromBNT162b2 vaccine recipients against N501 and Y501SARS-CoV-2.

Seven sera (indicated by triangles) were drawn 2 weeks after the seconddose of vaccine; 13 sera (indicated by circles) were drawn 4 weeks afterthe second dose.

FIG. 129 . Diagram of the N501Y substitution. L—leader sequence;ORF—open reading frame; RBD—receptor binding domain; S—spikeglycoprotein; S1—N-terminal furin cleavage fragment of S; S2—C-terminalfurin cleavage fragment of S; E—envelope protein; M—membrane protein;N—nucleoprotein; UTR—untranslated region.

FIG. 130 . Plaque morphologies of N501 and Y501 SARS-CoV-2 on Vero E6cells.

FIG. 131 . Scheme of the BNT162 vaccination and serum sampling.

FIG. 132 . Plot of the ratio of PRNT₅₀ between Y501 and N501 viruses.Triangles represent sera drawn two weeks after the second dose; circlesrepresent sera drawn four weeks after the second dose.

FIG. 133 . Engineered mutations.

Nucleotide and amino acid positions are indicated. Deletions aredepicted by dotted lines. Mutant nucleotides are in red. L, leadersequence; ORF, open reading frame; RBD, receptor binding domain; S,spike glycoprotein; S1, N-terminal furin cleavage fragment of S; S2,C-terminal furin cleavage fragment of S; E, envelope protein; M,membrane protein; N, nucleoprotein; UTR, untranslated region.

FIG. 134 . Plaque morphologies of WT (USA-WA1/2020), mutant N501Y,Δ69/70+N501Y+D614G, and E484K+N501Y+D614G SARS-CoV-2s on Vero E6 cells.

FIG. 135 . Scheme of the BNT162 vaccination and serum sampling.

FIG. 136 . PRNT₅₀s of twenty BNT162b2-vaccinated human sera againstwild-type (WT) and mutant SARS-CoV-2.

(a) WT (USA-WA1/2020) and mutant N501Y. (b) WT and Δ69/70+N501Y+D614G.(c) WT and E484K+N501Y+D614G. Seven (triangles) and thirteen (circles)sera were drawn 2 and 4 weeks after the second dose of vaccination,respectively. Sera with different PRNT₅₀s against WT and mutant virusesare connected by lines. Results in (a) were from one experiment; resultsin (b) and (c) were from another set of experiments. Each data point isthe average of duplicate assay results.

FIG. 137 . Ratios of neutralization GMTs against mutant viruses to GMTsagainst WT virus.

Triangles represent sera drawn two weeks after the second dose ofvaccination; circles represent sera drawn four weeks after the seconddose of vaccination.

FIG. 138 . Diagram of engineered spike substitutions and deletions.

The genome and sequence of clinical isolate USA-WA1/2020 are used as thewild-type virus in this study. Mutations from the United KingdomB.1.1.7, Brazilian P.1, and South African B.1.351 lineages arepresented. Deletions are indicated by dotted lines. Mutated nucleotidesare in red. Nucleotide and amino acid positions are indicated. L—leadersequence; ORF—open reading frame; RBD—receptor binding domain; S—spikeglycoprotein; S1—N-terminal furin cleavage fragment of S; S2—C-terminalfurin cleavage fragment of S; E—envelope protein; M—membrane protein;N—nucleoprotein; UTR—untranslated region.

FIG. 139 . Plaque morphologies of USA-WA1/2020 and mutant SARS-CoV-2's.

The plaque assays were performed on Vero E6 cells in 6-well plates.

FIG. 140 . Scheme of BNT162 immunization and serum collection.

FIG. 141 . Serum Neutralization of Variant Strains of SARS-CoV-2 afterthe Second Dose of BNT162b2 Vaccine.

Shown are the results of 50% plaque reduction neutralization testing(PRNT₅₀) with the use of 20 samples obtained from 15 trial participants2 weeks (circles) or 4 weeks (triangles) after the administration of thesecond dose of the BNT162b2 vaccine. The mutant viruses were obtained byengineering the full set of mutations in the B.1.1.7, P.1., or B.1.351lineages or subsets of the S gene mutations in the B.1.351 lineage(B.1.351−Δ242−244+D614G and B.1.351−RBD−D614G) into USA-WA1/2020. Eachdata point represents the geometric mean PRNT₅₀ obtained with a serumsample against the indicated virus, including data from repeatexperiments, as detailed in Table 31. The data for USA-WA1/2020 are fromthree experiments; for B.1.1.7-spike, B.1.351−Δ242−244+D614G, andB.1.351−RBD−D614G viruses from one experiment each; and for P.1-spikeand B.1.351-spike viruses from two experiments each. In each experiment,the neutralization titer was determined in duplicate assays, and thegeometric mean was taken. LOD: limit of detection.

FIG. 142 . Durability of BNT162b2-induced T cell responses.

PBMCs obtained on Day 1 (pre-prime), Day 29, Day 85, and Day 184 (7days, 9 and 23 weeks post-boost, respectively), were analyzed in ex vivoIFNγ ELISpot (for details see GA-RB-022-01A). Common pathogen T-cellepitope pools CEF (CMV, EBV, and influenza virus HLA class I epitopes)and CEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class IIepitopes) served to assess general T-cell reactivity, cell culturemedium served as negative control. Each dot represents the sum ofnormalized mean spot count from duplicate wells stimulated with twopeptide pools corresponding to the full-length wt S protein for onestudy subject, after subtraction of the medium-only control. Ratiosabove post-vaccination data points are the number of subjects withdetectable CD4⁺ or CD8⁺ T-cell responses within the total number oftested subjects per dose cohort and time-point.

FIG. 143 . A specific vaccine mRNA signal (red) is detected in the LN 6hpost injection using modV9 probe in dual IHC-ISH assay. Vaccine ismostly localized to subcapsular sinus (LN in 9 and 5 positions) and Bcell follicles (LN in 12 and 1 positions). Dendritic cells arevisualized by CD11c staining (turquoise, upper images) and only some ofthem uptake the vaccine. Majority of CD169+ macrophages (subcapsularsinus macrophages, turquoise, middle images) are positive for thevaccine. B cells (CD19+, turquoise, lower images) are the second majorpopulation showing vaccine signal.

FIG. 144 . A specific vaccine mRNA signal (red) is detected in thespleen 6h post injection using modV9 probe in dual IHC-ISH assay.Majority of the vaccine signal is detected in the white pulp. Dendriticcells are visualized by CD11c staining (turquoise, upper images) andonly some of them uptake the vaccine. A small portion of F4/80+macrophages (turquoise, middle images) uptake the vaccine. B cells(CD19+, turquoise, lower images) are the major population showing thevaccine signal.

FIG. 145 . Exemplary Stability Data.

Exemplary data from certain stability studies (see, for example, Example42, are shown for a BNT162b2 LNP preparation at indicated concentrationsand temperature conditions, as assessed by ELISA characterizingantibodies reactive to S1 spike protein.

DESCRIPTION OF THE SEQUENCES

The following table provides a listing of certain sequences referencedherein.

TABLE 1 DESCRIPTION OF THE SEQUENCES SEQ ID NO: Description SEQUENCEAntigenic S protein sequences  1 S protein (aminoMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKacid)SNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQ1LPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGINTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPINYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT  2 S protein (CDS)auguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggaguguauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaauucaugugucuggaacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauuagaggauggauuuuuggaacaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguuuuguaaugauccuuuucugggaguguauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuugaauaugugucucagccuuuucugauggaucuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaaauuuauucuaaacacacaccaauuaauuuagugagagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuacaagauuucagacacugcuggcucugcacagaucuuaucugacaccuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuaucugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuacagaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagccaacagaaucuauugugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaaaauaaaugugugaauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcagauaccacagaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaaucagguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaauguguuucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuaucagacacagacaaauuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucuaauaauucuauugcuauuccaacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauuuguggagauucuacagaauguucuaaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaaaaauacacaggaaguguuugcucaggugaaacagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuucuaaaccuucuaaaagaucuuuuauugaagaucugcuguuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugcaagagaucugauuugugcucagaaauuuaauggacugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuuacuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucugcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagugacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggauucucugucuucuacagcuucugcucugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggcaauuucuucugugcugaaugauauucugucuagacuggauaaaguggaagcugaagugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugugacacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuuguggaaaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcaccagcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuuaugaaccucagauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacuggauucuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucagaaagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugguacauuuggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggauguuguucuuguggaagcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca  3S protein RBDMFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR(aminoQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGacid)(V05) YQPYRVVVLSFELLHAPATVCGPK  4 S protein RBDauguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaag(CDS) (V05)auuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaa  5 S protein RBD/MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRFibritin (aminoQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGacid) (V05)YQPYRVVVLSFELLHAPATVCGPKGSPGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGauguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaa 6 S protein RBD/aauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucugFibritin (CDS)uacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauug(V05)uuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaggcucccccggcuccggcuccggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggaggugcuguuccagggccccggc  7 S protein PPMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEK(amino acid)SNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFV(V08/V09)FKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGINTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPINYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT  8 S protein PPauguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggagug(CDS) (V08)uauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaauucaugugucuggaacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauuagaggauggauuuuuggaacaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguuuuguaaugauccuuuucugggaguguauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuugaauaugugucucagccuuuucugauggaucuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaaauuuauucuaaacacacaccaauuaauuuagugagagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuacaagauuucagacacugcuggcucugcacagaucuuaucugacaccuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuaucugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuacagaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagccaacagaaucuauugugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaaaauaaaugugugaauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcagauaccacagaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaaucagguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaauguguuucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuaucagacacagacaaauuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucuaauaauucuauugcuauuccaacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauuuguggagauucuacagaauguucuaaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaaaaauacacaggaaguguuugcucaggugaaacagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuucuaaaccuucuaaaagaucuuuuauugaagaucugcuguuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugcaagagaucugauuugugcucagaaauuuaauggacugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuuacuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucugcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagugacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggauucucugucuucuacagcuucugcucugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggcaauuucuucugugcugaaugauauucugucuagacuggauccuccugaagcugaagugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugugacacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuuguggaaaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcaccagcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuuaugaaccucagauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacuggauucuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucagaaagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugguacauuuggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggauguuguucuuguggaagcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca  9S protein PPauguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaccaccagaacacagcugccuccagccuacaccaacagcuuuaccagaggcguguac(CDS) (V09)uaccccgacaagguguucagauccagcgugcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccugggcgucuacuaccacaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucgugcgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuucggcgagguguucaaugccaccagauucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuccgccagcuucagcaccuucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggagaugaagugcggcagauugccccuggacagacaggcaagaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaaccuggacuccaaagucggcggcaacuacaauuaccuguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcagcaccccuuguaacggcguggaaggcuucaacugcuacuucccacugcaguccuacggcuuucagcccacaaauggcgugggcuaucagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccaggacgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgagcacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaacagcccucggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaauagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaggacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccagaaugcccaggcacugaacacccuggucaagcagcuguccuccaacuucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacaca Foldon 10 Foldon (aminoGSGYIPEAPRDGQAYVRKDGEINVLLSTFLGRSLEVLFQGPG acid) 11 Foldon (CDS)ggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggaggugcuguuccagggccccggc 5'-UTR (hAg-Kozak) 12 5′-UTRAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC 3'-UTR (FI element) 133′-UTRCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC A30L70 14 A30L70AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

DETAILED DESCRIPTION

Although the present disclosure is described in detail below, it is tobe understood that this disclosure is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present disclosure which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present disclosure will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

In the following, the elements of the present disclosure will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and embodiments should not be construed to limit the presentdisclosure to only the explicitly described embodiments. Thisdescription should be understood to disclose and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed elements. Furthermore, any permutations and combinationsof all described elements should be considered disclosed by thisdescription unless the context indicates otherwise.

The term “about” means approximately or nearly, and in the context of anumerical value or range set forth herein in one embodiment means±20%,±10%, ±5%, or ±3% of the numerical value or range recited or claimed.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the disclosure (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wasindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the disclosure and does not pose alimitation on the scope of the claims. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the disclosure.

Unless expressly specified otherwise, the term “comprising” is used inthe context of the present document to indicate that further members mayoptionally be present in addition to the members of the list introducedby “comprising”. It is, however, contemplated as a specific embodimentof the present disclosure that the term “comprising” encompasses thepossibility of no further members being present, i.e., for the purposeof this embodiment “comprising” is to be understood as having themeaning of “consisting of” or “consisting essentially of”. Severaldocuments are cited throughout the text of this specification. Each ofthe documents cited herein (including all patents, patent applications,scientific publications, manufacturer's specifications, instructions,etc.), whether supra or infra, are hereby incorporated by reference intheir entirety. Nothing herein is to be construed as an admission thatthe present disclosure was not entitled to antedate such disclosure.

Definitions

In the following, definitions will be provided which apply to allaspects of the present disclosure. The following terms have thefollowing meanings unless otherwise indicated. Any undefined terms havetheir art recognized meanings.

Terms such as “reduce”, “decrease”, “inhibit” or “impair” as used hereinrelate to an overall reduction or the ability to cause an overallreduction, preferably of at least 5%, at least 10%, at least 20%, atleast 50%, at least 75% or even more, in the level. These terms includea complete or essentially complete inhibition, i.e., a reduction to zeroor essentially to zero.

Terms such as “increase”, “enhance” or “exceed” preferably relate to anincrease or enhancement by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 80%, at least 100%, at least 200%, atleast 500%, or even more.

According to the disclosure, the term “peptide” comprises oligo- andpolypeptides and refers to substances which comprise about two or more,about 3 or more, about 4 or more, about 6 or more, about 8 or more,about 10 or more, about 13 or more, about 16 or more, about 20 or more,and up to about 50, about 100 or about 150, consecutive amino acidslinked to one another via peptide bonds. The term “protein” or“polypeptide” refers to large peptides, in particular peptides having atleast about 150 amino acids, but the terms “peptide”, “protein” and“polypeptide” are used herein usually as synonyms.

A “therapeutic protein” has a positive or advantageous effect on acondition or disease state of a subject when provided to the subject ina therapeutically effective amount. In one embodiment, a therapeuticprotein has curative or palliative properties and may be administered toameliorate, relieve, alleviate, reverse, delay onset of or lessen theseverity of one or more symptoms of a disease or disorder. A therapeuticprotein may have prophylactic properties and may be used to delay theonset of a disease or to lessen the severity of such disease orpathological condition. The term “therapeutic protein” includes entireproteins or peptides, and can also refer to therapeutically activefragments thereof. It can also include therapeutically active variantsof a protein. Examples of therapeutically active proteins include, butare not limited to, antigens for vaccination and immunostimulants suchas cytokines.

“Fragment”, with reference to an amino acid sequence (peptide orprotein), relates to a part of an amino acid sequence, i.e. a sequencewhich represents the amino acid sequence shortened at the N-terminusand/or C-terminus. A fragment shortened at the C-terminus (N-terminalfragment) is obtainable e.g. by translation of a truncated open readingframe that lacks the 3′-end of the open reading frame. A fragmentshortened at the N-terminus (C-terminal fragment) is obtainable e.g. bytranslation of a truncated open reading frame that lacks the 5′-end ofthe open reading frame, as long as the truncated open reading framecomprises a start codon that serves to initiate translation. A fragmentof an amino acid sequence comprises e.g. at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% of the amino acid residues from anamino acid sequence. A fragment of an amino acid sequence preferablycomprises at least 6, in particular at least 8, at least 12, at least15, at least 20, at least 30, at least 50, or at least 100 consecutiveamino acids from an amino acid sequence.

By “variant” herein is meant an amino acid sequence that differs from aparent amino acid sequence by virtue of at least one amino acidmodification. The parent amino acid sequence may be a naturallyoccurring or wild type (WT) amino acid sequence, or may be a modifiedversion of a wild type amino acid sequence. Preferably, the variantamino acid sequence has at least one amino acid modification compared tothe parent amino acid sequence, e.g., from 1 to about 20 amino acidmodifications, and preferably from 1 to about 10 or from 1 to about 5amino acid modifications compared to the parent.

By “wild type” or “WT” or “native” herein is meant an amino acidsequence that is found in nature, including allelic variations. A wildtype amino acid sequence, peptide or protein has an amino acid sequencethat has not been intentionally modified.

For the purposes of the present disclosure, “variants” of an amino acidsequence (peptide, protein or polypeptide) comprise amino acid insertionvariants, amino acid addition variants, amino acid deletion variantsand/or amino acid substitution variants. The term “variant” includes allmutants, splice variants, posttranslationally modified variants,conformations, isoforms, allelic variants, species variants, and specieshomologs, in particularthose which are naturally occurring. The term“variant” includes, in particular, fragments of an amino acid sequence.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible. Amino acid addition variants comprise amino-and/or carboxy-terminal fusions of one or more amino acids, such as 1,2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletionvariants are characterized by the removal of one or more amino acidsfrom the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, ormore amino acids. The deletions may be in any position of the protein.Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpeptides and/or to replacing amino acids with other ones having similarproperties. Preferably, amino acid changes in peptide and proteinvariants are conservative amino acid changes, i.e., substitutions ofsimilarly charged or uncharged amino acids. A conservative amino acidchange involves substitution of one of a family of amino acids which arerelated in their side chains. Naturally occurring amino acids aregenerally divided into four families: acidic (aspartate, glutamate),basic (lysine, arginine, histidine), non-polar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),and uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine) amino acids. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Inone embodiment, conservative amino acid substitutions includesubstitutions within the following groups:

glycine, alanine;valine, isoleucine, leucine;aspartic acid, glutamic acid;asparagine, glutamine;serine, threonine;lysine, arginine; andphenylalanine, tyrosine.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 70%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, in some embodiments continuous amino acids. In someembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences. “Sequence identity” between two nucleic acid sequencesindicates the percentage of nucleotides that are identical between thesequences.

The terms “% identical”, “% identity” or similar terms are intended torefer, in particular, to the percentage of nucleotides or amino acidswhich are identical in an optimal alignment between the sequences to becompared. Said percentage is purely statistical, and the differencesbetween the two sequences may be but are not necessarily randomlydistributed over the entire length of the sequences to be compared.Comparisons of two sequences are usually carried out by comparing thesequences, after optimal alignment, with respect to a segment or “windowof comparison”, in order to identify local regions of correspondingsequences. The optimal alignment for a comparison may be carried outmanually or with the aid of the local homology algorithm by Smith andWaterman, 1981, Ads App. Math. 2, 482, with the aid of the localhomology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443,with the aid of the similarity search algorithm by Pearson and Lipman,1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computerprograms using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST Nand TFASTA in Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.). In some embodiments, percentidentity of two sequences is determined using the BLASTN or BLASTPalgorithm, as available on the United States National Center forBiotechnology Information (NCBI) website (e.g., atblast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for BLASTN algorithmon the NCBI website include: (i) Expect Threshold set to 10; (ii) WordSize set to 28; (iii) Max matches in a query range set to 0; (iv)Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and(vi) the filter for low complexity regions being used. In someembodiments, the algorithm parameters used for BLASTP algorithm on theNCBI website include: (i) Expect Threshold set to 10; (ii) Word Size setto 3; (iii) Max matches in a query range set to 0; (iv) Matrix set toBLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi)conditional compositional score matrix adjustment.

Percentage identity is obtained by determining the number of identicalpositions at which the sequences to be compared correspond, dividingthis number by the number of positions compared (e.g., the number ofpositions in the reference sequence) and multiplying this result by 100.

In some embodiments, the degree of similarity or identity is given for aregion which is at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or about 100% of the entirelength of the reference sequence. For example, if the reference nucleicacid sequence consists of 200 nucleotides, the degree of identity isgiven for at least about 100, at least about 120, at least about 140, atleast about 160, at least about 180, or about 200 nucleotides, in someembodiments continuous nucleotides. In some embodiments, the degree ofsimilarity or identity is given for the entire length of the referencesequence. Homologous amino acid sequences exhibit according to thedisclosure at least 40%, in particular at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% and preferably at least 95%, atleast 98 or at least 99% identity of the amino acid residues.

The amino acid sequence variants described herein may readily beprepared by the skilled person, for example, by recombinant DNAmanipulation. The manipulation of DNA sequences for preparing peptidesor proteins having substitutions, additions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example. Furthermore,the peptides and amino acid variants described herein may be readilyprepared with the aid of known peptide synthesis techniques such as, forexample, by solid phase synthesis and similar methods.

In one embodiment, a fragment or variant of an amino acid sequence(peptide or protein) is preferably a “functional fragment” or“functional variant”. The term “functional fragment” or “functionalvariant” of an amino acid sequence relates to any fragment or variantexhibiting one or more functional properties identical or similar tothose of the amino acid sequence from which it is derived, i.e., it isfunctionally equivalent. With respect to antigens or antigenicsequences, one particular function is one or more immunogenic activitiesdisplayed by the amino acid sequence from which the fragment or variantis derived. The term “functional fragment” or “functional variant”, asused herein, in particular refers to a variant molecule or sequence thatcomprises an amino acid sequence that is altered by one or more aminoacids compared to the amino acid sequence of the parent molecule orsequence and that is still capable of fulfilling one or more of thefunctions of the parent molecule or sequence, e.g., inducing an immuneresponse. In one embodiment, the modifications in the amino acidsequence of the parent molecule or sequence do not significantly affector alter the characteristics of the molecule or sequence. In differentembodiments, the function of the functional fragment or functionalvariant may be reduced but still significantly present, e.g.,immunogenicity of the functional variant may be at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of the parent moleculeor sequence. However, in other embodiments, immunogenicity of thefunctional fragment or functional variant may be enhanced compared tothe parent molecule or sequence.

An amino acid sequence (peptide, protein or polypeptide) “derived from”a designated amino acid sequence (peptide, protein or polypeptide)refers to the origin of the first amino acid sequence. Preferably, theamino acid sequence which is derived from a particular amino acidsequence has an amino acid sequence that is identical, essentiallyidentical or homologous to that particular sequence or a fragmentthereof. Amino acid sequences derived from a particular amino acidsequence may be variants of that particular sequence or a fragmentthereof. For example, it will be understood by one of ordinary skill inthe art that the antigens suitable for use herein may be altered suchthat they vary in sequence from the naturally occurring or nativesequences from which they were derived, while retaining the desirableactivity of the native sequences.

As used herein, an “instructional material” or “instructions” includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositions andmethods of the invention. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thecompositions of the invention or be shipped together with a containerwhich contains the compositions. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compositions be usedcooperatively by the recipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated”, but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated”. An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant object”such as a recombinant nucleic acid in the context of the presentinvention is not occurring naturally.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

“Physiological pH” as used herein refers to a pH of about 7.5.

The term “genetic modification” or simply “modification” includes thetransfection of cells with nucleic acid. The term “transfection” relatesto the introduction of nucleic acids, in particular RNA, into a cell.For purposes of the present invention, the term “transfection” alsoincludes the introduction of a nucleic acid into a cell or the uptake ofa nucleic acid by such cell, wherein the cell may be present in asubject, e.g., a patient. Thus, according to the present invention, acell for transfection of a nucleic acid described herein can be presentin vitro or in vivo, e.g. the cell can form part of an organ, a tissueand/or an organism of a patient. According to the invention,transfection can be transient or stable. For some applications oftransfection, it is sufficient if the transfected genetic material isonly transiently expressed. RNA can be transfected into cells totransiently express its coded protein. Since the nucleic acid introducedin the transfection process is usually not integrated into the nucleargenome, the foreign nucleic acid will be diluted through mitosis ordegraded. Cells allowing episomal amplification of nucleic acids greatlyreduce the rate of dilution. If it is desired that the transfectednucleic acid actually remains in the genome of the cell and its daughtercells, a stable transfection must occur. Such stable transfection can beachieved by using virus-based systems or transposon-based systems fortransfection. Generally, nucleic acid encoding antigen is transientlytransfected into cells. RNA can be transfected into cells to transientlyexpress its coded protein.

The term “seroconversion” includes a ≥4-fold rise from beforevaccination to 1-month post Dose 2.

Coronavirus

Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNAviruses and are phylogenetically divided into four genera (α, β, γ, andδ), with betacoronaviruses further subdivided into four lineages (A, B,C, and D). Coronaviruses infect a wide range of avian and mammalianspecies, including humans. Some human coronaviruses generally cause mildrespiratory diseases, although severity can be greater in infants, theelderly, and the immunocompromised. Middle East respiratory syndromecoronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus(SARS-CoV), belonging to betacoronavirus lineages C and B, respectively,are highly pathogenic. Both viruses emerged into the human populationfrom animal reservoirs within the last 15 years and caused outbreakswith high case-fatality rates. The outbreak of severe acute respiratorysyndrome coronavirus-2 (SARS-CoV-2) that causes atypical pneumonia(coronavirus disease 2019; COVID-19) has raged in China sincemid-December 2019, and has developed to be a public health emergency ofinternational concern. SARS-CoV-2 (MN908947.3) belongs tobetacoronavirus lineage B. It has at least 70% sequence similarity toSARS-CoV.

In general, coronaviruses have four structural proteins, namely,envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and Mproteins have important functions in the viral assembly, and the Nprotein is necessary for viral RNA synthesis. The critical glycoproteinS is responsible for virus binding and entry into target cells. The Sprotein is synthesized as a single-chain inactive precursor that iscleaved by furin-like host proteases in the producing cell into twononcovalently associated subunits, S1 and S2. The S1 subunit containsthe receptor-binding domain (RBD), which recognizes the host-cellreceptor. The S2 subunit contains the fusion peptide, two heptadrepeats, and a transmembrane domain, all of which are required tomediate fusion of the viral and host-cell membranes by undergoing alarge conformational rearrangement. The S1 and S2 subunits trimerize toform a large prefusion spike.

The S precursor protein of SARS-CoV-2 can be proteolytically cleavedinto S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit consists ofthe receptor-binding domain (RBD), which mediates virus entry intosensitive cells through the host angiotensin-converting enzyme 2 (ACE2)receptor.

Antigen

The present invention comprises the use of RNA encoding an amino acidsequence comprising SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof. Thus, the RNA encodes a peptide or proteincomprising at least an epitope SARS-CoV-2 S protein or an immunogenicvariant thereof for inducing an immune response against coronavirus Sprotein, in particular SARS-CoV-2 S protein in a subject. The amino acidsequence comprising SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof (i.e., the antigenic peptide or protein) isalso designated herein as “vaccine antigen”, “peptide and proteinantigen”, “antigen molecule” or simply “antigen”. The SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is alsodesignated herein as “antigenic peptide or protein” or “antigenicsequence”.

SARS-CoV-2 coronavirus full length spike (S) protein consist of 1273amino acids (see SEQ ID NO: 1). In specific embodiments, full lengthspike (S) protein according to SEQ ID NO: 1 is modified in such a waythat the prototypical prefusion conformation is stabilized.Stabilization of the prefusion conformation may be obtained byintroducing two consecutive proline substitutions at AS residues 986 and987 in the full length spike protein. Specifically, spike (S) proteinstabilized protein variants are obtained in a way that the amino acidresidue at position 986 is exchanged to proline and the amino acidresidue at position 987 is also exchanged to proline. In one embodiment,a SARS-CoV-2 S protein variant comprises the amino acid sequence shownin SEQ ID NO: 7.

In one embodiment, the vaccine antigen described herein comprises,consists essentially of or consists of a spike protein (S) ofSARS-CoV-2, a variant thereof, or a fragment thereof.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7,or an immunogenic fragment of the amino acid sequence of amino acids 17to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acidsequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In oneembodiment, a vaccine antigen comprises the amino acid sequence of aminoacids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 17 to 1273 ofSEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 1273 of SEQ IDNO: 1 or 7. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, the vaccine antigen comprises, consists essentiallyof or consists of SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of aspike protein (S) of SARS-CoV-2), a variant thereof, or a fragmentthereof.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 17 to 683of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 683 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 17 to 683 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 683 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 17 to 683 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 17 to 685of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 17 to 685 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 685 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1.

In one embodiment, the vaccine antigen comprises, consists essentiallyof or consists of the receptor binding domain (RBD) of the S1 subunit ofa spike protein (S) of SARS-CoV-2, a variant thereof, or a fragmentthereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, a variant thereof, or a fragment thereof is also referred to hereinas “RBD” or “RBD domain”.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 327 to 528 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 327 to 528 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9,a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 327 to 528 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 327 to 528 of SEQ ID NO: 1.

According to certain embodiments, a signal peptide is fused, eitherdirectly or through a linker, to a SARS-CoV-2 S protein, a variantthereof, or a fragment thereof, i.e., the antigenic peptide or protein.Accordingly, in one embodiment, a signal peptide is fused to the abovedescribed amino acid sequences derived from SARS-CoV-2 S protein orimmunogenic fragments thereof (antigenic peptides or proteins) comprisedby the vaccine antigens described above.

Such signal peptides are sequences, which typically exhibit a length ofabout 15 to 30 amino acids and are preferably located at the N-terminusof the antigenic peptide or protein, without being limited thereto.Signal peptides as defined herein preferably allow the transport of theantigenic peptide or protein as encoded by the RNA into a definedcellular compartment, preferably the cell surface, the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. In oneembodiment, the signal peptide sequence as defined herein includes,without being limited thereto, the signal peptide sequence of SARS-CoV-2S protein, in particular a sequence comprising the amino acid sequenceof amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or a functionalvariant thereof.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to16 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or afunctional fragment of the amino acid sequence of amino acids 1 to 16 ofSEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 1 to 16 of SEQ ID NO: 1. In one embodiment, RNA encoding a signalsequence (i) comprises the nucleotide sequence of nucleotides 1 to 48 ofSEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to19 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or afunctional fragment of the amino acid sequence of amino acids 1 to 19 ofSEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 1 to 19 of SEQ ID NO: 1. In one embodiment, RNA encoding a signalsequence (i) comprises the nucleotide sequence of nucleotides 1 to 57 ofSEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1.

The signal peptide sequence as defined herein further includes, withoutbeing limited thereto, the signal peptide sequence of an immunoglobulin,e.g., the signal peptide sequence of an immunoglobulin heavy chainvariable region, wherein the immunoglobulin may be human immunoglobulin.In particular, the signal peptide sequence as defined herein includes asequence comprising the amino acid sequence of amino acids 1 to 22 ofSEQ ID NO: 31 or a functional variant thereof.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functionalfragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to22 of SEQ ID NO: 31. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 119 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 119 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 22 of SEQ ID NO: 31, or a functional fragment of theamino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQID NO: 31. In one embodiment, RNA encoding a signal sequence (i)comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 22 of SEQ ID NO: 31.

Such signal peptides are preferably used in order to promote secretionof the encoded antigenic peptide or protein. More preferably, a signalpeptide as defined herein is fused to an encoded antigenic peptide orprotein as defined herein.

Accordingly, in particularly preferred embodiments, the RNA describedherein comprises at least one coding region encoding an antigenicpeptide or protein and a signal peptide, said signal peptide preferablybeing fused to the antigenic peptide or protein, more preferably to theN-terminus of the antigenic peptide or protein as described herein.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofSEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 1 or 7. In one embodiment, a vaccine antigen comprises theamino acid sequence of SEQ ID NO: 1 or 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a fragment of thenucleotide sequence of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 1or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1 or 7. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 1or 7.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 7, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20,24, or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 15,16, 19, 20, 24, or 25, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 7, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 7, or animmunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 7. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenicfragment of the amino acid sequence of amino acids 1 to 683 of SEQ IDNO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids1 to 683 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprisesthe amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1 to 683 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 1 to 683 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 1 to 683 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenicfragment of the amino acid sequence of amino acids 1 to 685 of SEQ IDNO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids1 to 685 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprisesthe amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1 to 685 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 1 to 685 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 685 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 1 to 685 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 3, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 3.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 4, or a fragment of the nucleotidesequence of SEQ ID NO: 4, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 3, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 3, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 3.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 221of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 221 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 221 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 716 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 716 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 221 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 221 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 221 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 221 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 224of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 224 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 224 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 725 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 725 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 224 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 224 of SEQ ID NO: 31.

According to certain embodiments, a trimerization domain is fused,either directly or through a linker, e.g., a glycine/serine linker, to aSARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e.,the antigenic peptide or protein. Accordingly, in one embodiment, atrimerization domain is fused to the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above (which may optionally be fused to a signalpeptide as described above).

Such trimerization domains are preferably located at the C-terminus ofthe antigenic peptide or protein, without being limited thereto.Trimerization domains as defined herein preferably allow thetrimerization of the antigenic peptide or protein as encoded by the RNA.Examples of trimerization domains as defined herein include, withoutbeing limited thereto, foldon, the natural trimerization domain of T4fibritin. The C-terminal domain of T4 fibritin (foldon) is obligatoryfor the formation of the fibritin trimer structure and can be used as anartificial trimerization domain. In one embodiment, the trimerizationdomain as defined herein includes, without being limited thereto, asequence comprising the amino acid sequence of amino acids 3 to 29 ofSEQ ID NO: 10 or a functional variant thereof. In one embodiment, thetrimerization domain as defined herein includes, without being limitedthereto, a sequence comprising the amino acid sequence of SEQ ID NO: 10or a functional variant thereof.

In one embodiment, a trimerization domain comprises the amino acidsequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or afunctional fragment of the amino acid sequence of amino acids 3 to 29 ofSEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 3 to 29 of SEQ ID NO: 10. In one embodiment, a trimerizationdomain comprises the amino acid sequence of amino acids 3 to 29 of SEQID NO: 10.

In one embodiment, RNA encoding a trimerization domain (i) comprises thenucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQID NO: 11, or a fragment of the nucleotide sequence of nucleotides 7 to87 of SEQ ID NO: 11, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of amino acids 3 to 29of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the aminoacid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO:10. In one embodiment, RNA encoding a trimerization domain (i) comprisesthe nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof amino acids 3 to 29 of SEQ ID NO: 10.

In one embodiment, a trimerization domain comprises the amino acidsequence SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofSEQ ID NO: 10, or a functional fragment of the amino acid sequence ofSEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 10. In one embodiment, a trimerization domain comprises the aminoacid sequence of SEQ ID NO: 10.

In one embodiment, RNA encoding a trimerization domain (i) comprises thenucleotide sequence of SEQ ID NO: 11, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 11, or a fragment of the nucleotidesequence of SEQ ID NO: 11, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 10, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 10, or a functionalfragment of the amino acid sequence of SEQ ID NO: 10, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment,RNA encoding a trimerization domain (i) comprises the nucleotidesequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 10.

Such trimerization domains are preferably used in order to promotetrimerization of the encoded antigenic peptide or protein. Morepreferably, a trimerization domain as defined herein is fused to anantigenic peptide or protein as defined herein.

Accordingly, in particularly preferred embodiments, the RNA describedherein comprises at least one coding region encoding an antigenicpeptide or protein and a trimerization domain as defined herein, saidtrimerization domain preferably being fused to the antigenic peptide orprotein, more preferably to the C-terminus of the antigenic peptide orprotein.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 5, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotidesequence of SEQ ID NO: 6, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 5, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 5, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 17, 21, or 26, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment ofthe nucleotide sequence of SEQ ID NO: 17, 21, or 26, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 5, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 18, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 18, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:18. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 18.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 257of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 257 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 257 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 824 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 257 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 257 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 260of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 260 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 260 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 833 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 260 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 260 of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 20 to 257of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 20 to 257 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 20 to 257 ofSEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 111 to 824 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides111 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 20 to 257of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ IDNO: 30; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 20 to 257 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 23 to 260of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 23 to 260 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 23 to 260 ofSEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 120 to 833 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides120 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 23 to 260of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ IDNO: 32; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

According to certain embodiments, a transmembrane domain is fused,either directly or through a linker, e.g., a glycine/serine linker, to aSARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e.,the antigenic peptide or protein. Accordingly, in one embodiment, atransmembrane domain is fused to the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above (which may optionally be fused to a signalpeptide and/or trimerization domain as described above).

Such transmembrane domains are preferably located at the C-terminus ofthe antigenic peptide or protein, without being limited thereto.Preferably, such transmembrane domains are located at the C-terminus ofthe trimerization domain, if present, without being limited thereto. Inone embodiment, a trimerization domain is present between the SARS-CoV-2S protein, a variant thereof, or a fragment thereof, i.e., the antigenicpeptide or protein, and the transmembrane domain.

Transmembrane domains as defined herein preferably allow the anchoringinto a cellular membrane of the antigenic peptide or protein as encodedby the RNA.

In one embodiment, the transmembrane domain sequence as defined hereinincludes, without being limited thereto, the transmembrane domainsequence of SARS-CoV-2 S protein, in particular a sequence comprisingthe amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or afunctional variant thereof.

In one embodiment, a transmembrane domain sequence comprises the aminoacid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1207 to 1254 of SEQID NO: 1, or a functional fragment of the amino acid sequence of aminoacids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In oneembodiment, a transmembrane domain sequence comprises the amino acidsequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

In one embodiment, RNA encoding a transmembrane domain sequence (i)comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ IDNO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides3619 to 3762 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotidesequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or thenucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1207 to 1254 of SEQ IDNO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acidsequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1207 to 1254 of SEQID NO: 1. In one embodiment, RNA encoding a transmembrane domainsequence (i) comprises the nucleotide sequence of nucleotides 3619 to3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1207 to 1254 of SEQ IDNO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 311of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 311 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 311 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 986 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 311 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 311 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 314of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 314 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 314 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 995 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 314 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 314 of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 20 to 311of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 20 to 311 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 20 to 311 ofSEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 111 to 986 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides111 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 20 to 311of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ IDNO: 30; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 20 to 311 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 23 to 314of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 23 to 314 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 23 to 314 ofSEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 120 to 995 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides120 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 23 to 314of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ IDNO: 32; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 23 to 314 of SEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 30, or a fragment of the nucleotidesequence of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 29, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 29, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 29, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 32, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 32, or a fragment of the nucleotidesequence of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 31, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 31, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 31, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 31. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 28, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 28.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 27, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 27, or a fragment of the nucleotidesequence of SEQ ID NO: 27, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 28, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 28, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 28. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 28.

In one embodiment, the vaccine antigens described above comprise acontiguous sequence of SARS-CoV-2 coronavirus spike (S) protein thatconsists of or essentially consists of the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above. In one embodiment, the vaccine antigensdescribed above comprise a contiguous sequence of SARS-CoV-2 coronavirusspike (S) protein of no more than 220 amino acids, 215 amino acids, 210amino acids, or 205 amino acids.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2(RBP020.1 or RBP020.2). In one embodiment, RNA encoding a vaccineantigen is nucleoside modified messenger RNA (modRNA) described hereinas RBP020.2.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 5. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 19, or 20, a nucleotide sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ IDNO: 19, or 20, and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 7, or an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of SEQ ID NO: 7. In one embodiment, RNA encoding a vaccineantigen is nucleoside modified messenger RNA (modRNA) and (i) comprisesthe nucleotide sequence of SEQ ID NO: 19, or 20; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 7. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 7.

As used herein, the term “vaccine” refers to a composition that inducesan immune response upon inoculation into a subject. In some embodiments,the induced immune response provides protective immunity.

In one embodiment, the RNA encoding the antigen molecule is expressed incells of the subject to provide the antigen molecule. In one embodiment,expression of the antigen molecule is at the cell surface or into theextracellular space. In one embodiment, the antigen molecule ispresented in the context of MHC. In one embodiment, the RNA encoding theantigen molecule is transiently expressed in cells of the subject. Inone embodiment, after administration of the RNA encoding the antigenmolecule, in particular after intramuscular administration of the RNAencoding the antigen molecule, expression of the RNA encoding theantigen molecule in muscle occurs. In one embodiment, afteradministration of the RNA encoding the antigen molecule, expression ofthe RNA encoding the antigen molecule in spleen occurs. In oneembodiment, after administration of the RNA encoding the antigenmolecule, expression of the RNA encoding the antigen molecule in antigenpresenting cells, preferably professional antigen presenting cellsoccurs. In one embodiment, the antigen presenting cells are selectedfrom the group consisting of dendritic cells, macrophages and B cells.In one embodiment, after administration of the RNA encoding the antigenmolecule, no or essentially no expression of the RNA encoding theantigen molecule in lung and/or liver occurs. In one embodiment, afteradministration of the RNA encoding the antigen molecule, expression ofthe RNA encoding the antigen molecule in spleen is at least 5-fold theamount of expression in lung.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to lymph nodes and/or spleen. In someembodiments, RNA encoding a vaccine antigen is detectable in lymph nodesand/or spleen 6 hours or later following administration and preferablyup to 6 days or longer.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to B cell follicles, subcapsular sinus,and/or T cell zone, in particular B cell follicles and/or subcapsularsinus of lymph nodes.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to B cells (CD19+), subcapsular sinusmacrophages (CD169+) and/or dendritic cells (CD11c+) in the T cell zoneand intermediary sinus of lymph nodes, in particular to B cells (CD19+)and/or subcapsular sinus macrophages (CD169+) of lymph nodes.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to white pulp of spleen.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to B cells, DCs (CD11c+), in particular thosesurrounding the B cells, and/or microphages of spleen, in particular toB cells and/or DCs (CD11c+).

In one embodiment, the vaccine antigen is expressed in lymph node and/orspleen, in particular in the cells of lymph node and/or spleen describedabove.

The peptide and protein antigens suitable for use according to thedisclosure typically include a peptide or protein comprising an epitopeof SARS-CoV-2 S protein or a functional variant thereof for inducing animmune response. The peptide or protein or epitope may be derived from atarget antigen, i.e. the antigen against which an immune response is tobe elicited. For example, the peptide or protein antigen or the epitopecontained within the peptide or protein antigen may be a target antigenor a fragment or variant of a target antigen. The target antigen may bea coronavirus S protein, in particular SARS-CoV-2 S protein.

The antigen molecule or a procession product thereof, e.g., a fragmentthereof, may bind to an antigen receptor such as a BCR or TCR carried byimmune effector cells, or to antibodies. A peptide and protein antigenwhich is provided to a subject according to the invention byadministering RNA encoding the peptide and protein antigen, i.e., avaccine antigen, preferably results in the induction of an immuneresponse, e.g., a humoral and/or cellular immune response in the subjectbeing provided the peptide or protein antigen. Said immune response ispreferably directed against a target antigen, in particular coronavirusS protein, in particular SARS-CoV-2 S protein. Thus, a vaccine antigenmay comprise the target antigen, a variant thereof, or a fragmentthereof. In one embodiment, such fragment or variant is immunologicallyequivalent to the target antigen. In the context of the presentdisclosure, the term “fragment of an antigen” or “variant of an antigen”means an agent which results in the induction of an immune responsewhich immune response targets the antigen, i.e. a target antigen. Thus,the vaccine antigen may correspond to or may comprise the targetantigen, may correspond to or may comprise a fragment of the targetantigen or may correspond to or may comprise an antigen which ishomologous to the target antigen or a fragment thereof. Thus, accordingto the disclosure, a vaccine antigen may comprise an immunogenicfragment of a target antigen or an amino acid sequence being homologousto an immunogenic fragment of a target antigen. An “immunogenic fragmentof an antigen” according to the disclosure preferably relates to afragment of an antigen which is capable of inducing an immune responseagainst the target antigen. The vaccine antigen may be a recombinantantigen.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect. Inthe context of the present disclosure, the term “immunologicallyequivalent” is preferably used with respect to the immunological effectsor properties of antigens or antigen variants used for immunization. Forexample, an amino acid sequence is immunologically equivalent to areference amino acid sequence if said amino acid sequence when exposedto the immune system of a subject induces an immune reaction having aspecificity of reacting with the reference amino acid sequence.

“Activation” or “stimulation”, as used herein, refers to the state of animmune effector cell such as T cell that has been sufficientlystimulated to induce detectable cellular proliferation. Activation canalso be associated with initiation of signaling pathways, inducedcytokine production, and detectable effector functions. The term“activated immune effector cells” refers to, among other things, immuneeffector cells that are undergoing cell division.

The term “priming” refers to a process wherein an immune effector cellsuch as a T cell has its first contact with its specific antigen andcauses differentiation into effector cells such as effector T cells.

The term “clonal expansion” or “expansion” refers to a process wherein aspecific entity is multiplied. In the context of the present disclosure,the term is preferably used in the context of an immunological responsein which immune effector cells are stimulated by an antigen,proliferate, and the specific immune effector cell recognizing saidantigen is amplified. Preferably, clonal expansion leads todifferentiation of the immune effector cells.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response can be generated. The term “antigen” includes,in particular, proteins and peptides. In one embodiment, an antigen ispresented by cells of the immune system such as antigen presenting cellslike dendritic cells or macrophages. An antigen or a procession productthereof such as a T-cell epitope is in one embodiment bound by a T- orB-cell receptor, or by an immunoglobulin molecule such as an antibody.Accordingly, an antigen or a procession product thereof may reactspecifically with antibodies or T lymphocytes (T cells). In oneembodiment, an antigen is a viral antigen, such as a coronavirus Sprotein, e.g., SARS-CoV-2 S protein, and an epitope is derived from suchantigen.

The term “viral antigen” refers to any viral component having antigenicproperties, i.e. being able to provoke an immune response in anindividual. The viral antigen may be coronavirus S protein, e.g.,SARS-CoV-2 S protein.

The term “expressed on the cell surface” or “associated with the cellsurface” means that a molecule such as an antigen is associated with andlocated at the plasma membrane of a cell, wherein at least a part of themolecule faces the extracellular space of said cell and is accessiblefrom the outside of said cell, e.g., by antibodies located outside thecell. In this context, a part is preferably at least 4, preferably atleast 8, preferably at least 12, more preferably at least 20 aminoacids. The association may be direct or indirect. For example, theassociation may be by one or more transmembrane domains, one or morelipid anchors, or by the interaction with any other protein, lipid,saccharide, or other structure that can be found on the outer leaflet ofthe plasma membrane of a cell. For example, a molecule associated withthe surface of a cell may be a transmembrane protein having anextracellular portion or may be a protein associated with the surface ofa cell by interacting with another protein that is a transmembraneprotein.

“Cell surface” or “surface of a cell” is used in accordance with itsnormal meaning in the art, and thus includes the outside of the cellwhich is accessible to binding by proteins and other molecules. Anantigen is expressed on the surface of cells if it is located at thesurface of said cells and is accessible to binding by e.g.antigen-specific antibodies added to the cells.

The term “extracellular portion” or “exodomain” in the context of thepresent invention refers to a part of a molecule such as a protein thatis facing the extracellular space of a cell and preferably is accessiblefrom the outside of said cell, e.g., by binding molecules such asantibodies located outside the cell. Preferably, the term refers to oneor more extracellular loops or domains or a fragment thereof.

The term “epitope” refers to a part or fragment of a molecule such as anantigen that is recognized by the immune system. For example, theepitope may be recognized by T cells, B cells or antibodies. An epitopeof an antigen may include a continuous or discontinuous portion of theantigen and may be between about 5 and about 100, such as between about5 and about 50, more preferably between about 8 and about 30, mostpreferably between about 8 and about 25 amino acids in length, forexample, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In oneembodiment, an epitope is between about 10 and about 25 amino acids inlength. The term “epitope” includes T cell epitopes.

The term “T cell epitope” refers to a part or fragment of a protein thatis recognized by a T cell when presented in the context of MHCmolecules. The term “major histocompatibility complex” and theabbreviation “MHC” includes MHC class I and MHC class II molecules andrelates to a complex of genes which is present in all vertebrates. MHCproteins or molecules are important for signaling between lymphocytesand antigen presenting cells or diseased cells in immune reactions,wherein the MHC proteins or molecules bind peptide epitopes and presentthem for recognition by T cell receptors on T cells. The proteinsencoded by the MHC are expressed on the surface of cells, and displayboth self-antigens (peptide fragments from the cell itself) andnon-self-antigens (e.g., fragments of invading microorganisms) to a Tcell. In the case of class I MHC/peptide complexes, the binding peptidesare typically about 8 to about 10 amino acids long although longer orshorter peptides may be effective. In the case of class II MHC/peptidecomplexes, the binding peptides are typically about 10 to about 25 aminoacids long and are in particular about 13 to about 18 amino acids long,whereas longer and shorter peptides may be effective.

The peptide and protein antigen can be 2-100 amino acids, including forexample, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids,25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 aminoacids, or 50 amino acids in length. In some embodiments, a peptide canbe greater than 50 amino acids. In some embodiments, the peptide can begreater than 100 amino acids.

The peptide or protein antigen can be any peptide or protein that caninduce or increase the ability of the immune system to developantibodies and T cell responses to the peptide or protein.

In one embodiment, vaccine antigen is recognized by an immune effectorcell. Preferably, the vaccine antigen if recognized by an immuneeffector cell is able to induce in the presence of appropriateco-stimulatory signals, stimulation, priming and/or expansion of theimmune effector cell carrying an antigen receptor recognizing thevaccine antigen. In the context of the embodiments of the presentinvention, the vaccine antigen is preferably presented or present on thesurface of a cell, preferably an antigen presenting cell. In oneembodiment, an antigen is presented by a diseased cell such as avirus-infected cell. In one embodiment, an antigen receptor is a TCRwhich binds to an epitope of an antigen presented in the context of MHC.In one embodiment, binding of a TCR when expressed by T cells and/orpresent on T cells to an antigen presented by cells such as antigenpresenting cells results in stimulation, priming and/or expansion ofsaid T cells. In one embodiment, binding of a TCR when expressed by Tcells and/or present on T cells to an antigen presented on diseasedcells results in cytolysis and/or apoptosis of the diseased cells,wherein said T cells preferably release cytotoxic factors, e.g.perforins and granzymes.

In one embodiment, an antigen receptor is an antibody or B cell receptorwhich binds to an epitope in an antigen. In one embodiment, an antibodyor B cell receptor binds to native epitopes of an antigen.

Nucleic Acids

The term “polynucleotide” or “nucleic acid”, as used herein, is intendedto include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantlyproduced and chemically synthesized molecules. A nucleic acid may besingle-stranded or double-stranded. RNA includes in vitro transcribedRNA (IVT RNA) or synthetic RNA. According to the invention, apolynucleotide is preferably isolated.

Nucleic acids may be comprised in a vector. The term “vector” as usedherein includes any vectors known to the skilled person includingplasmid vectors, cosmid vectors, phage vectors such as lambda phage,viral vectors such as retroviral, adenoviral or baculoviral vectors, orartificial chromosome vectors such as bacterial artificial chromosomes(BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes(PAC). Said vectors include expression as well as cloning vectors.Expression vectors comprise plasmids as well as viral vectors andgenerally contain a desired coding sequence and appropriate DNAsequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments.

In one embodiment of all aspects of the invention, the RNA encoding thevaccine antigen is expressed in cells such as antigen presenting cellsof the subject treated to provide the vaccine antigen.

The nucleic acids described herein may be recombinant and/or isolatedmolecules.

In the present disclosure, the term “RNA” relates to a nucleic acidmolecule which includes ribonucleotide residues. In preferredembodiments, the RNA contains all or a majority of ribonucleotideresidues. As used herein, “ribonucleotide” refers to a nucleotide with ahydroxyl group at the 2′-position of a β-D-ribofuranosyl group. RNAencompasses without limitation, double stranded RNA, single strandedRNA, isolated RNA such as partially purified RNA, essentially pure RNA,synthetic RNA, recombinantly produced RNA, as well as modified RNA thatdiffers from naturally occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations may refer to addition of non-nucleotide material to internalRNA nucleotides or to the end(s) of RNA. It is also contemplated hereinthat nucleotides in RNA may be non-standard nucleotides, such aschemically synthesized nucleotides or deoxynucleotides. For the presentdisclosure, these altered RNAs are considered analogs ofnaturally-occurring RNA.

In certain embodiments of the present disclosure, the RNA is messengerRNA (mRNA) that relates to a RNA transcript which encodes a peptide orprotein. As established in the art, mRNA generally contains a 5′untranslated region (5′-UTR), a peptide coding region and a 3′untranslated region (3′-UTR). In some embodiments, the RNA is producedby in vitro transcription or chemical synthesis. In one embodiment, themRNA is produced by in vitro transcription using a DNA template whereDNA refers to a nucleic acid that contains deoxyribonucleotides.

In one embodiment, RNA is in vitro transcribed RNA (IVT-RNA) and may beobtained by in vitro transcription of an appropriate DNA template. Thepromoter for controlling transcription can be any promoter for any RNApolymerase. A DNA template for in vitro transcription may be obtained bycloning of a nucleic acid, in particular cDNA, and introducing it intoan appropriate vector for in vitro transcription. The cDNA may beobtained by reverse transcription of RNA. In certain embodiments of thepresent disclosure, the RNA is “replicon RNA” or simply a “replicon”, inparticular “self-replicating RNA” or “self-amplifying RNA”. In oneparticularly preferred embodiment, the replicon or self-replicating RNAis derived from or comprises elements derived from a ssRNA virus, inparticular a positive-stranded ssRNA virus such as an alphavirus.Alphaviruses are typical representatives of positive-stranded RNAviruses. Alphaviruses replicate in the cytoplasm of infected cells (forreview of the alphaviral life cycle see Jose et al., Future Microbiol.,2009, vol. 4, pp. 837-856). The total genome length of many alphavirusestypically ranges between 11,000 and 12,000 nucleotides, and the genomicRNA typically has a 5′-cap, and a 3′ poly(A) tail. The genome ofalphaviruses encodes non-structural proteins (involved in transcription,modification and replication of viral RNA and in protein modification)and structural proteins (forming the virus particle). There aretypically two open reading frames (ORFs) in the genome. The fournon-structural proteins (nsP1-nsP4) are typically encoded together by afirst ORF beginning near the 5′ terminus of the genome, while alphavirusstructural proteins are encoded together by a second ORF which is founddownstream of the first ORF and extends near the 3′ terminus of thegenome. Typically, the first ORF is larger than the second ORF, theratio being roughly 2:1. In cells infected by an alphavirus, only thenucleic acid sequence encoding non-structural proteins is translatedfrom the genomic RNA, while the genetic information encoding structuralproteins is translatable from a subgenomic transcript, which is an RNAmolecule that resembles eukaryotic messenger RNA (mRNA; Gould et al.,2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. atearly stages of the viral life cycle, the (+) stranded genomic RNAdirectly acts like a messenger RNA for the translation of the openreading frame encoding the non-structural poly-protein (nsP1234).Alphavirus-derived vectors have been proposed for delivery of foreigngenetic information into target cells or target organisms. In simpleapproaches, the open reading frame encoding alphaviral structuralproteins is replaced by an open reading frame encoding a protein ofinterest. Alphavirus-based trans-replication systems rely on alphavirusnucleotide sequence elements on two separate nucleic acid molecules: onenucleic acid molecule encodes a viral replicase, and the other nucleicacid molecule is capable of being replicated by said replicase in trans(hence the designation trans-replication system). Trans-replicationrequires the presence of both these nucleic acid molecules in a givenhost cell. The nucleic acid molecule capable of being replicated by thereplicase in trans must comprise certain alphaviral sequence elements toallow recognition and RNA synthesis by the alphaviral replicase.

In one embodiment, the RNA described herein may have modifiednucleosides. In some embodiments, the RNA comprises a modifiednucleoside in place of at least one (e.g., every) uridine.

The term “uracil,” as used herein, describes one of the nucleobases thatcan occur in the nucleic acid of RNA. The structure of uracil is:

The term “uridine,” as used herein, describes one of the nucleosidesthat can occur in RNA. The structure of uridine is:

UTP (uridine 5′-triphosphate) has the following structure:

Pseudo-UTP (pseudouridine 5′-triphosphate) has the following structure:

“Pseudouridine” is one example of a modified nucleoside that is anisomer of uridine, where the uracil is attached to the pentose ring viaa carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m14W),which has the structure:

N1-methyl-pseudo-UTP has the following structure:

Another exemplary modified nucleoside is 5-methyl-uridine (m5U), whichhas the structure:

In some embodiments, one or more uridine in the RNA described herein isreplaced by a modified nucleoside. In some embodiments, the modifiednucleoside is a modified uridine.

In some embodiments, RNA comprises a modified nucleoside in place of atleast one uridine.

In some embodiments, RNA comprises a modified nucleoside in place ofeach uridine.

In some embodiments, the modified nucleoside is independently selectedfrom pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and5-methyl-uridine (m5U). In some embodiments, the modified nucleosidecomprises pseudouridine (ψ). In some embodiments, the modifiednucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments,the modified nucleoside comprises 5-methyl-uridine (m5U). In someembodiments, RNA may comprise more than one type of modified nucleoside,and the modified nucleosides are independently selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U). In some embodiments, the modified nucleosides comprisepseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In someembodiments, the modified nucleosides comprise pseudouridine (ψ) and5-methyl-uridine (m5U). In some embodiments, the modified nucleosidescomprise N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). Insome embodiments, the modified nucleosides comprise pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In some embodiments, the modified nucleoside replacing one or more,e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine(m³U), 5-methoxy-uridine (mo⁵U), 5-aza-uridine, 6-aza-uridine,2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U),4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U),5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyaceticacid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine(mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm5s²U),1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine,5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Urn), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or anyother modified uridine known in the art.

In one embodiment, the RNA comprises other modified nucleosides orcomprises further modified nucleosides, e.g., modified cytidine. Forexample, in one embodiment, in the RNA 5-methylcytidine is substitutedpartially or completely, preferably completely, for cytidine. In oneembodiment, the RNA comprises 5-methylcytidine and one or more selectedfrom pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and5-methyl-uridine (m5U). In one embodiment, the RNA comprises5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments,the RNA comprises 5-methylcytidine in place of each cytidine andN1-methyl-pseudouridine (m1ψ) in place of each uridine.

In some embodiments, the RNA according to the present disclosurecomprises a 5′-cap. In one embodiment, the RNA of the present disclosuredoes not have uncapped 5′-triphosphates. In one embodiment, the RNA maybe modified by a 5′-cap analog. The term “5′-cap” refers to a structurefound on the 5′-end of an mRNA molecule and generally consists of aguanosine nucleotide connected to the mRNA via a 5′- to 5′-triphosphatelinkage. In one embodiment, this guanosine is methylated at the7-position. Providing an RNA with a 5′-cap or 5′-cap analog may beachieved by in vitro transcription, in which the 5′-cap isco-transcriptionally expressed into the RNA strand, or may be attachedto RNA post-transcriptionally using capping enzymes. In someembodiments, the building block cap for RNA is m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG (also sometimes referred to as m₂^(7,3′O)G(5′)ppp(5′)m^(2′-O)ApG), which has the following structure:

Below is an exemplary Cap1 RNA, which comprises RNA and m₂^(7,3′O)G(5′)ppp(5′)m^(2′-O)ApG:

Below is another exemplary Cap1 RNA (no cap analog):

In some embodiments, the RNA is modified with “Cap0” structures using,in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m₂^(7,3′O)G(5′)ppp(5′)G)) with the structure:

Below is an exemplary Cap0 RNA comprising RNA and m₂^(7,3′O)G(5′)ppp(5′)G:

In some embodiments, the “Cap0” structures are generated using the capanalog Beta-S-ARCA (m₂ ^(7,2′O)G(5′)ppSp(5′)G) with the structure:

Below is an exemplary Cap0 RNA comprising Beta-S-ARCA (m₂^(7,2′O)G(5′)ppSp(5′)G) and RNA:

The “D1” diastereomer of beta-S-ARCA or “beta-S-ARCA(D1)” is thediastereomer of beta-S-ARCA which elutes first on an HPLC columncompared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) andthus exhibits a shorter retention time (cf., WO 2011/015347, hereinincorporated by reference).

A particularly preferred cap is beta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG) orm₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG. In some embodiments, RNA according tothe present disclosure comprises a 5′-UTR and/or a 3′-UTR. The term“untranslated region” or “UTR” relates to a region in a DNA moleculewhich is transcribed but is not translated into an amino acid sequence,or to the corresponding region in an RNA molecule, such as an mRNAmolecule. An untranslated region (UTR) can be present 5′ (upstream) ofan open reading frame (5′-UTR) and/or 3′ (downstream) of an open readingframe (3′-UTR). A 5′-UTR, if present, is located at the 5′ end, upstreamof the start codon of a protein-encoding region. A 5′-UTR is downstreamof the 5′-cap (if present), e.g. directly adjacent to the 5′-cap. A3′-UTR, if present, is located at the 3′ end, downstream of thetermination codon of a protein-encoding region, but the term “3′-UTR”does preferably not include the poly(A) sequence. Thus, the 3′-UTR isupstream of the poly(A) sequence (if present), e.g. directly adjacent tothe poly(A) sequence.

In some embodiments, RNA comprises a 5′-UTR comprising the nucleotidesequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 12.

In some embodiments, RNA comprises a 3′-UTR comprising the nucleotidesequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 13.

A particularly preferred 5′-UTR comprises the nucleotide sequence of SEQID NO: 12. A particularly preferred 3′-UTR comprises the nucleotidesequence of SEQ ID NO: 13.

In some embodiments, the RNA according to the present disclosurecomprises a 3′-poly(A) sequence.

As used herein, the term “poly(A) sequence” or “poly-A tail” refers toan uninterrupted or interrupted sequence of adenylate residues which istypically located at the 3′-end of an RNA molecule. Poly(A) sequencesare known to those of skill in the art and may follow the 3′-UTR in theRNAs described herein. An uninterrupted poly(A) sequence ischaracterized by consecutive adenylate residues. In nature, anuninterrupted poly(A) sequence is typical. RNAs disclosed herein canhave a poly(A) sequence attached to the free 3′-end of the RNA by atemplate-independent RNA polymerase after transcription or a poly(A)sequence encoded by DNA and transcribed by a template-dependent RNApolymerase.

It has been demonstrated that a poly(A) sequence of about 120 Anucleotides has a beneficial influence on the levels of RNA intransfected eukaryotic cells, as well as on the levels of protein thatis translated from an open reading frame that is present upstream (5′)of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp.4009-4017).

The poly(A) sequence may be of any length. In some embodiments, apoly(A) sequence comprises, essentially consists of, or consists of atleast 20, at least 30, at least 40, at least 80, or at least 100 and upto 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides,and, in particular, about 120 A nucleotides. In this context,“essentially consists of” means that most nucleotides in the poly(A)sequence, typically at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% by number of nucleotides in the poly(A) sequence are A nucleotides,but permits that remaining nucleotides are nucleotides other than Anucleotides, such as U nucleotides (uridylate), G nucleotides(guanylate), or C nucleotides (cytidylate). In this context, “consistsof” means that all nucleotides in the poly(A) sequence, i.e., 100% bynumber of nucleotides in the poly(A) sequence, are A nucleotides. Theterm “A nucleotide” or “A” refers to adenylate.

In some embodiments, a poly(A) sequence is attached during RNAtranscription, e.g., during preparation of in vitro transcribed RNA,based on a DNA template comprising repeated dT nucleotides(deoxythymidylate) in the strand complementary to the coding strand. TheDNA sequence encoding a poly(A) sequence (coding strand) is referred toas poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strandof DNA essentially consists of dA nucleotides, but is interrupted by arandom sequence of the four nucleotides (dA, dC, dG, and dT). Suchrandom sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides inlength. Such a cassette is disclosed in WO 2016/005324 A1, herebyincorporated by reference. Any poly(A) cassette disclosed in WO2016/005324 A1 may be used in the present invention. A poly(A) cassettethat essentially consists of dA nucleotides, but is interrupted by arandom sequence having an equal distribution of the four nucleotides(dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows,on DNA level, constant propagation of plasmid DNA in E. coli and isstill associated, on RNA level, with the beneficial properties withrespect to supporting RNA stability and translational efficiency isencompassed. Consequently, in some embodiments, the poly(A) sequencecontained in an RNA molecule described herein essentially consists of Anucleotides, but is interrupted by a random sequence of the fournucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30,or 10 to 20 nucleotides in length.

In some embodiments, no nucleotides other than A nucleotides flank apoly(A) sequence at its 3′-end, i.e., the poly(A) sequence is not maskedor followed at its 3′-end by a nucleotide other than A.

In some embodiments, the poly(A) sequence may comprise at least 20, atleast 30, at least 40, at least 80, or at least 100 and up to 500, up to400, up to 300, up to 200, or up to 150 nucleotides. In someembodiments, the poly(A) sequence may essentially consist of at least20, at least 30, at least 40, at least 80, or at least 100 and up to500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In someembodiments, the poly(A) sequence may consist of at least 20, at least30, at least 40, at least 80, or at least 100 and up to 500, up to 400,up to 300, up to 200, or up to 150 nucleotides. In some embodiments, thepoly(A) sequence comprises at least 100 nucleotides. In someembodiments, the poly(A) sequence comprises about 150 nucleotides. Insome embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, RNA comprises a poly(A) sequence comprising thenucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 14. A particularly preferred poly(A)sequence comprises the nucleotide sequence of SEQ ID NO: 14.

According to the disclosure, vaccine antigen is preferably administeredas single-stranded, 5′-capped mRNA that is translated into therespective protein upon entering cells of a subject being administeredthe RNA. Preferably, the RNA contains structural elements optimized formaximal efficacy of the RNA with respect to stability and translationalefficiency (5′-cap, 5′-UTR, 3′-UTR, poly(A) sequence).

In one embodiment, beta-S-ARCA(D1) is utilized as specific cappingstructure at the 5′-end of the RNA. In one embodiment, m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG is utilized as specific capping structureat the 5′-end of the RNA. In one embodiment, the 5′-UTR sequence isderived from the human alpha-globin mRNA and optionally has an optimized‘Kozak sequence’ to increase translational efficiency. In oneembodiment, a combination of two sequence elements (FI element) derivedfrom the “amino terminal enhancer of split” (AES) mRNA (called F) andthe mitochondrial encoded 12S ribosomal RNA (called I) are placedbetween the coding sequence and the poly(A) sequence to assure highermaximum protein levels and prolonged persistence of the mRNA. In oneembodiment, two re-iterated 3′-UTRs derived from the human beta-globinmRNA are placed between the coding sequence and the poly(A) sequence toassure higher maximum protein levels and prolonged persistence of themRNA. In one embodiment, a poly(A) sequence measuring 110 nucleotides inlength, consisting of a stretch of 30 adenosine residues, followed by a10 nucleotide linker sequence and another 70 adenosine residues is used.This poly(A) sequence was designed to enhance RNA stability andtranslational efficiency.

In one embodiment of all aspects of the invention, RNA encoding avaccine antigen is expressed in cells of the subject treated to providethe vaccine antigen. In one embodiment of all aspects of the invention,the RNA is transiently expressed in cells of the subject. In oneembodiment of all aspects of the invention, the RNA is in vitrotranscribed RNA. In one embodiment of all aspects of the invention,expression of the vaccine antigen is at the cell surface. In oneembodiment of all aspects of the invention, the vaccine antigen isexpressed and presented in the context of MHC. In one embodiment of allaspects of the invention, expression of the vaccine antigen is into theextracellular space, i.e., the vaccine antigen is secreted.

In the context of the present disclosure, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intopeptide or protein.

According to the present invention, the term “transcription” comprises“in vitro transcription”, wherein the term “in vitro transcription”relates to a process wherein RNA, in particular mRNA, is in vitrosynthesized in a cell-free system, preferably using appropriate cellextracts. Preferably, cloning vectors are applied for the generation oftranscripts. These cloning vectors are generally designated astranscription vectors and are according to the present inventionencompassed by the term “vector”. According to the present invention,the RNA used in the present invention preferably is in vitro transcribedRNA (IVT-RNA) and may be obtained by in vitro transcription of anappropriate DNA template. The promoter for controlling transcription canbe any promoter for any RNA polymerase. Particular examples of RNApolymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the invitro transcription according to the invention is controlled by a T7 orSP6 promoter. A DNA template for in vitro transcription may be obtainedby cloning of a nucleic acid, in particular cDNA, and introducing itinto an appropriate vector for in vitro transcription. The cDNA may beobtained by reverse transcription of RNA.

With respect to RNA, the term “expression” or “translation” relates tothe process in the ribosomes of a cell by which a strand of mRNA directsthe assembly of a sequence of amino acids to make a peptide or protein.

In one embodiment, after administration of the RNA described herein,e.g., formulated as RNA lipid particles, at least a portion of the RNAis delivered to a target cell. In one embodiment, at least a portion ofthe RNA is delivered to the cytosol of the target cell. In oneembodiment, the RNA is translated by the target cell to produce thepeptide or protein it enodes. In one embodiment, the target cell is aspleen cell. In one embodiment, the target cell is an antigen presentingcell such as a professional antigen presenting cell in the spleen. Inone embodiment, the target cell is a dendritic cell or macrophage. RNAparticles such as RNA lipid particles described herein may be used fordelivering RNA to such target cell. Accordingly, the present disclosurealso relates to a method for delivering RNA to a target cell in asubject comprising the administration of the RNA particles describedherein to the subject. In one embodiment, the RNA is delivered to thecytosol of the target cell. In one embodiment, the RNA is translated bythe target cell to produce the peptide or protein encoded by the RNA.“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

In one embodiment, the RNA encoding vaccine antigen to be administeredaccording to the invention is non-immunogenic. RNA encodingimmunostimulant may be administered according to the invention toprovide an adjuvant effect. The RNA encoding immunostimulant may bestandard RNA or non-immunogenic RNA.

The term “non-immunogenic RNA” as used herein refers to RNA that doesnot induce a response by the immune system upon administration, e.g., toa mammal, or induces a weaker response than would have been induced bythe same RNA that differs only in that it has not been subjected to themodifications and treatments that render the non-immunogenic RNAnon-immunogenic, i.e., than would have been induced by standard RNA(stdRNA). In one preferred embodiment, non-immunogenic RNA, which isalso termed modified RNA (modRNA) herein, is rendered non-immunogenic byincorporating modified nucleosides suppressing RNA-mediated activationof innate immune receptors into the RNA and removing double-stranded RNA(dsRNA).

For rendering the non-immunogenic RNA non-immunogenic by theincorporation of modified nucleosides, any modified nucleoside may beused as long as it lowers or suppresses immunogenicity of the RNA.Particularly preferred are modified nucleosides that suppressRNA-mediated activation of innate immune receptors. In one embodiment,the modified nucleosides comprises a replacement of one or more uridineswith a nucleoside comprising a modified nucleobase. In one embodiment,the modified nucleobase is a modified uracil. In one embodiment, thenucleoside comprising a modified nucleobase is selected from the groupconsisting of 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U),5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U),uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine(cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine(chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine(mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(m⁵s²U),1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine,5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine. Inone particularly preferred embodiment, the nucleoside comprising amodified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ)or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine.

In one embodiment, the replacement of one or more uridines with anucleoside comprising a modified nucleobase comprises a replacement ofat least 1%, at least 2%, at least 3%, at least 4%, at least 5%, atleast 10%, at least 25%, at least 50%, at least 75%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% of the uridines.

During synthesis of mRNA by in vitro transcription (IVT) using T7 RNApolymerase significant amounts of aberrant products, includingdouble-stranded RNA (dsRNA) are produced due to unconventional activityof the enzyme. dsRNA induces inflammatory cytokines and activateseffector enzymes leading to protein synthesis inhibition. dsRNA can beremoved from RNA such as IVT RNA, for example, by ion-pair reversedphase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene(PS-DVB) matrix. Alternatively, an enzymatic based method using E. coliRNaseIII that specifically hydrolyzes dsRNA but not ssRNA, therebyeliminating dsRNA contaminants from IVT RNA preparations can be used.Furthermore, dsRNA can be separated from ssRNA by using a cellulosematerial. In one embodiment, an RNA preparation is contacted with acellulose material and the ssRNA is separated from the cellulosematerial under conditions which allow binding of dsRNA to the cellulosematerial and do not allow binding of ssRNA to the cellulose material.

As the term is used herein, “remove” or “removal” refers to thecharacteristic of a population of first substances, such asnon-immunogenic RNA, being separated from the proximity of a populationof second substances, such as dsRNA, wherein the population of firstsubstances is not necessarily devoid of the second substance, and thepopulation of second substances is not necessarily devoid of the firstsubstance. However, a population of first substances characterized bythe removal of a population of second substances has a measurably lowercontent of second substances as compared to the non-separated mixture offirst and second substances.

In one embodiment, the removal of dsRNA from non-immunogenic RNAcomprises a removal of dsRNA such that less than 10%, less than 5%, lessthan 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, lessthan 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNAcomposition is dsRNA. In one embodiment, the non-immunogenic RNA is freeor essentially free of dsRNA. In some embodiments, the non-immunogenicRNA composition comprises a purified preparation of single-strandednucleoside modified RNA. For example, in some embodiments, the purifiedpreparation of single-stranded nucleoside modified RNA is substantiallyfree of double stranded RNA (dsRNA). In some embodiments, the purifiedpreparation is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or at least 99.9% single stranded nucleosidemodified RNA, relative to all other nucleic acid molecules (DNA, dsRNA,etc.).

In one embodiment, the non-immunogenic RNA is translated in a cell moreefficiently than standard RNA with the same sequence. In one embodiment,translation is enhanced by a factor of 2-fold relative to its unmodifiedcounterpart. In one embodiment, translation is enhanced by a 3-foldfactor. In one embodiment, translation is enhanced by a 4-fold factor.In one embodiment, translation is enhanced by a 5-fold factor. In oneembodiment, translation is enhanced by a 6-fold factor. In oneembodiment, translation is enhanced by a 7-fold factor. In oneembodiment, translation is enhanced by an 8-fold factor. In oneembodiment, translation is enhanced by a 9-fold factor. In oneembodiment, translation is enhanced by a 10-fold factor. In oneembodiment, translation is enhanced by a 15-fold factor. In oneembodiment, translation is enhanced by a 20-fold factor. In oneembodiment, translation is enhanced by a 50-fold factor. In oneembodiment, translation is enhanced by a 100-fold factor. In oneembodiment, translation is enhanced by a 200-fold factor. In oneembodiment, translation is enhanced by a 500-fold factor. In oneembodiment, translation is enhanced by a 1000-fold factor. In oneembodiment, translation is enhanced by a 2000-fold factor. In oneembodiment, the factor is 10-1000-fold. In one embodiment, the factor is10-100-fold. In one embodiment, the factor is 10-200-fold. In oneembodiment, the factor is 10-300-fold. In one embodiment, the factor is10-500-fold. In one embodiment, the factor is 20-1000-fold. In oneembodiment, the factor is 30-1000-fold. In one embodiment, the factor is50-1000-fold. In one embodiment, the factor is 100-1000-fold. In oneembodiment, the factor is 200-1000-fold. In one embodiment, translationis enhanced by any other significant amount or range of amounts.

In one embodiment, the non-immunogenic RNA exhibits significantly lessinnate immunogenicity than standard RNA with the same sequence. In oneembodiment, the non-immunogenic RNA exhibits an innate immune responsethat is 2-fold less than its unmodified counterpart. In one embodiment,innate immunogenicity is reduced by a 3-fold factor. In one embodiment,innate immunogenicity is reduced by a 4-fold factor. In one embodiment,innate immunogenicity is reduced by a 5-fold factor. In one embodiment,innate immunogenicity is reduced by a 6-fold factor. In one embodiment,innate immunogenicity is reduced by a 7-fold factor. In one embodiment,innate immunogenicity is reduced by a 8-fold factor. In one embodiment,innate immunogenicity is reduced by a 9-fold factor. In one embodiment,innate immunogenicity is reduced by a 10-fold factor. In one embodiment,innate immunogenicity is reduced by a 15-fold factor. In one embodiment,innate immunogenicity is reduced by a 20-fold factor. In one embodiment,innate immunogenicity is reduced by a 50-fold factor. In one embodiment,innate immunogenicity is reduced by a 100-fold factor. In oneembodiment, innate immunogenicity is reduced by a 200-fold factor. Inone embodiment, innate immunogenicity is reduced by a 500-fold factor.In one embodiment, innate immunogenicity is reduced by a 1000-foldfactor. In one embodiment, innate immunogenicity is reduced by a2000-fold factor.

The term “exhibits significantly less innate immunogenicity” refers to adetectable decrease in innate immunogenicity. In one embodiment, theterm refers to a decrease such that an effective amount of thenon-immunogenic RNA can be administered without triggering a detectableinnate immune response. In one embodiment, the term refers to a decreasesuch that the non-immunogenic RNA can be repeatedly administered withouteliciting an innate immune response sufficient to detectably reduceproduction of the protein encoded by the non-immunogenic RNA. In oneembodiment, the decrease is such that the non-immunogenic RNA can berepeatedly administered without eliciting an innate immune responsesufficient to eliminate detectable production of the protein encoded bythe non-immunogenic RNA. “Immunogenicity” is the ability of a foreignsubstance, such as RNA, to provoke an immune response in the body of ahuman or other animal. The innate immune system is the component of theimmune system that is relatively unspecific and immediate. It is one oftwo main components of the vertebrate immune system, along with theadaptive immune system.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence.

As used herein, the terms “linked,” “fused”, or “fusion” are usedinterchangeably. These terms refer to the joining together of two ormore elements or components or domains.

Codon-Optimization/Increase in G/C Content

In some embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof describedherein is encoded by a coding sequence which is codon-optimized and/orthe G/C content of which is increased compared to wild type codingsequence. This also includes embodiments, wherein one or more sequenceregions of the coding sequence are codon-optimized and/or increased inthe G/C content compared to the corresponding sequence regions of thewild type coding sequence. In one embodiment, the codon-optimizationand/or the increase in the G/C content preferably does not change thesequence of the encoded amino acid sequence.

The term “codon-optimized” refers to the alteration of codons in thecoding region of a nucleic acid molecule to reflect the typical codonusage of a host organism without preferably altering the amino acidsequence encoded by the nucleic acid molecule. Within the context of thepresent invention, coding regions are preferably codon-optimized foroptimal expression in a subject to be treated using the RNA moleculesdescribed herein. Codon-optimization is based on the finding that thetranslation efficiency is also determined by a different frequency inthe occurrence of tRNAs in cells. Thus, the sequence of RNA may bemodified such that codons for which frequently occurring tRNAs areavailable are inserted in place of “rare codons”.

In some embodiments of the invention, the guanosine/cytosine (G/C)content of the coding region of the RNA described herein is increasedcompared to the G/C content of the corresponding coding sequence of thewild type RNA, wherein the amino acid sequence encoded by the RNA ispreferably not modified compared to the amino acid sequence encoded bythe wild type RNA. This modification of the RNA sequence is based on thefact that the sequence of any RNA region to be translated is importantfor efficient translation of that mRNA. Sequences having an increased G(guanosine)/C (cytosine) content are more stable than sequences havingan increased A (adenosine)/U (uracil) content. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).Depending on the amino acid to be encoded by the RNA, there are variouspossibilities for modification of the RNA sequence, compared to its wildtype sequence. In particular, codons which contain A and/or Unucleotides can be modified by substituting these codons by othercodons, which code for the same amino acids but contain no A and/or U orcontain a lower content of A and/or U nucleotides.

In various embodiments, the G/C content of the coding region of the RNAdescribed herein is increased by at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 55%, or even more compared tothe G/C content of the coding region of the wild type RNA.

Embodiments of Administered RNAs

In some embodiments, compositions or medical preparations describedherein comprise RNA encoding an amino acid sequence comprisingSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.Likewise, methods described herein comprise administration of such RNA.

The active platform for use herein is based on an antigen-coding RNAvaccine to induce robust neutralising antibodies andaccompanying/concomitant T cell response to achieve protectiveimmunization with preferably minimal vaccine doses. The RNA administeredis preferably in-vitro transcribed RNA.

Three different RNA platforms are particularly preferred, namelynon-modified uridine containing mRNA (uRNA), nucleoside modified mRNA(modRNA) and self-amplifying RNA (saRNA). In one particularly preferredembodiment, the RNA is in vitro transcribed RNA.

As described herein, embodiments of each of these platforms are assessedherein (see, for example Example 2), representing a novel and powerfulapproach to and system for rapid vaccine development. This describedapproach and system achieved remarkable and efficient success, enablingdevelopment of an effective clinical candidate within several months ofprovision of antigen (e.g., SARS-CoV-2 S1 protein and/or RBD thereof)sequence (as described herein, relevant sequence information (e.g.,GenBank: MN908947.3) became available in January 2020). Insights andadvantages embodied in this described approach and system include, forexample, ability to directly compare one or more features of differentstrategies to achieve rapid, efficient, and effective development. Amongother things, the present disclosure encompasses insights that identifythe source of a problem with more typical strategies for vaccinedevelopment. Moreover, findings included herein establish a variety ofadvantages and benefits, particularly in rapid vaccine development andnotably of special benefit in a pandemic.

As described herein, in some embodiments, vaccine candidates areassessed for titer of antibodies induced in a model organism (e.g.,mouse; see e.g., Example 2) directed to an encoded antigen (e.g., S1protein) or portion thereof (e.g., RBD). In some embodiments, vaccinecandidates are assessed for pseudoviral neutralization (see e.g.,Example 2) activity of induced antibodies. In some embodiments, vaccinecandidates are characterized for nature of T cell response induced(e.g., T_(H)1 vs T_(H)2 character; see, e.g., Example 4). In someembodiments, vaccine candidates are assessed in more than one modelorganism (see. E.g., Examples 2, Example 4, etc)

In the following, embodiments of these three different RNA platforms aredescribed, wherein certain terms used when describing elements thereofhave the following meanings:

S152 protein/S1S2 RBD: Sequences encoding the respective antigen ofSARS-CoV-2.nsP1, nsP2, nsP3, and nsP4: Wildtype sequences encoding the Venezuelanequine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicaseand a subgenomic promotor plus conserved sequence elements supportingreplication and translation.virUTR: Viral untranslated region encoding parts of the subgenomicpromotor as well as replication and translation supporting sequenceelements.hAg-Kozak: 5′-UTR sequence of the human alpha-globin mRNA with anoptimized ‘Kozak sequence’ to increase translational efficiency.Sec: Sec corresponds to the intrinsic S1S2 protein secretory signalpeptide (sec), which guides translocation of the nascent polypeptidechain into the endoplasmatic reticulum.Glycine-serine linker (GS): Sequences coding for short linker peptidespredominantly consisting of the amino acids glycine (G) and serine (S),as commonly used for fusion proteins.Fibritin: Partial sequence of T4 fibritin (foldon), used as artificialtrimerization domain.TM: TM sequence corresponds to the transmembrane part of the S1S2protein.FI element: The 3′-UTR is a combination of two sequence elements derivedfrom the “amino terminal enhancer of split” (AES) mRNA (called F) andthe mitochondrial encoded 12S ribosomal RNA (called I). These wereidentified by an ex vivo selection process for sequences that confer RNAstability and augment total protein expression.A30L70: A poly(A)-tail measuring 110 nucleotides in length, consistingof a stretch of 30 adenosine residues, followed by a 10 nucleotidelinker sequence and another 70 adenosine residues designed to enhanceRNA stability and translational efficiency in dendritic cells.

In general, vaccine RNA described herein may comprise, from 5′ to 3′,one of the following structures:

-   -   Cap-5′-UTR-Vaccine Antigen-Encoding Sequence-3′-UTR-Poly(A)        or    -   beta-S-ARCA(D1)-hAg-Kozak-Vaccine Antigen-Encoding        Sequence-FI-A30L70.

In general, a vaccine antigen described herein may comprise, fromN-terminus to C-terminus, one of the following structures:

-   -   Signal Sequence-RBD-Trimerization Domain        or    -   Signal Sequence-RBD-Trimerization Domain-Transmembrane Domain.

RBD and Trimerization Domain may be separated by a linker, in particulara GS linker such as a linker having the amino acid sequence GSPGSGSGS(SEQ ID NO: 33). Trimerization Domain and Transmembrane Domain may beseparated by a linker, in particular a GS linker such as a linker havingthe amino acid sequence GSGSGS (SEQ ID NO: 34).

Signal Sequence may be a signal sequence as described herein. RBD may bea RBD domain as described herein. Trimerization Domain may be atrimerization domain as described herein. Transmembrane Domain may be atransmembrane domain as described herein.

In one embodiment,

-   -   Signal sequence comprises the amino acid sequence of amino acids        1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of        amino acids 1 to 22 of SEQ ID NO: 31, or an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to this amino acid sequence,    -   RBD comprises the amino acid sequence of amino acids 327 to 528        of SEQ ID NO: 1, or an amino acid sequence having at least 99%,        98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid        sequence,    -   Trimerization Domain comprises the amino acid sequence of amino        acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ        ID NO: 10, or an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid        sequence; and    -   Transmembrane Domain comprises the amino acid sequence of amino        acids 1207 to 1254 of SEQ ID NO: 1, or an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to this amino acid sequence.

In one embodiment,

-   -   Signal sequence comprises the amino acid sequence of amino acids        1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of        amino acids 1 to 22 of SEQ ID NO: 31,    -   RBD comprises the amino acid sequence of amino acids 327 to 528        of SEQ ID NO: 1,    -   Trimerization Domain comprises the amino acid sequence of amino        acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ        ID NO: 10; and    -   Transmembrane Domain comprises the amino acid sequence of amino        acids 1207 to 1254 of SEQ ID NO: 1.

The above described RNA or RNA encoding the above described vaccineantigen may be non-modified uridine containing mRNA (uRNA), nucleosidemodified mRNA (modRNA) or self-amplifying RNA (saRNA). In oneembodiment, the above described RNA or RNA encoding the above describedvaccine antigen is nucleoside modified mRNA (modRNA).

Non-Modified Uridine Messenger RNA (uRNA)

The active principle of the non-modified messenger RNA (uRNA) drugsubstance is a single-stranded mRNA that is translated upon entering acell. In addition to the sequence encoding the coronavirus vaccineantigen (i.e. open reading frame), each uRNA preferably contains commonstructural elements optimized for maximal efficacy of the RNA withrespect to stability and translational efficiency (5′-cap, 5′-UTR,3′-UTR, poly(A)-tail). The preferred 5′ cap structure is beta-S-ARCA(D1)(m₂ ^(7,2′-O)GppSpG). The preferred 5′-UTR and 3′-UTR comprise thenucleotide sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQID NO: 13, respectively. The preferred poly(A)-tail comprises thesequence of SEQ ID NO: 14.

Different embodiment of this platform are as follows:

RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded Viral spike protein(S1S2 protein) of the SARS-CoV-2 antigen (S1S2 full-length protein,sequence variant) RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded Viral spike protein(S1S2 protein) of the SARS-CoV-2 antigen (S1S2 full-length protein,sequence variant) BNT162a1; RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5)Structure beta-S-ARCA(D1)-hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 EncodedViral spike protein (S protein) of the SARS-CoV-2 (partial antigensequence, Receptor Binding Domain (RBD) of S1S2 protein)

FIG. 19 schematizes the general structure of the antigen-encoding RNAs.

Nucleotide Sequence of RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40         50 52GGGCGAACUA GUAUUCUUCU GGUCCCCACA GACUCAGAGA GAACCCGCCA CC                           hAg-Kozak        62         72         82         92        102        112AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein       122        132        142        152        162        172AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein       182        192        202        212        222        232AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein       242        252        262        272        282        292AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein       302        312        322        332        342        352AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein       362        372        382        392        402        412AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein       422        432        442        452        462        472AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein       482        492        502        512        522        532CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein       542        552        562        572        582        592UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein       602        612        622        632        642        652GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein       662        672        682        692        702        712UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein       722        732        742        752        762        772UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein       782        792        802        812        822        832CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein       842        852        862        872        882        892GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein       902        912        922        932        942        952GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein       962        972        982        992       1002       1012UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      1022       1032       1042       1052       1062       1072CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      1082       1092       1102       1112       1122       1132GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      1142       1152       1162       1172       1182       1192UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      1202       1212       1222       1232       1242       1252GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      1262       1272       1282       1292       1302       1312GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      1322       1332       1342       1352       1362       1372UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      1382       1392       1402       1412       1422       1432UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      1442       1452       1462       1472       1482       1492CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      1502       1512       1522       1532       1542       1552AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      1562       1572       1582       1592       1602       1612AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      1622       1632       1642       1652       1662       1672CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      1682       1692       1702       1712       1722       1732UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      1742       1752       1762       1772       1782       1792CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      1802       1812       1822       1832       1842       1852ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      1862       1872       1882       1892       1902       1912GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      1922       1932       1942       1952       1962       1972CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      1982       1992       2002       2012       2022       2032AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      2042       2052       2062       2072       2082       2092GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      2102       2112       2122       2132       2142       2152CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      2162       2172       2182       2192       2202       2212GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      2222       2232       2242       2252       2262       2272UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      2282       2292       2302       2312       2322       2332UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      2342       2352       2362       2372       2382       2392ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      2402       2412       2422       2432       2442       2452GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      2462       2472       2482       2492       2502       2512AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein      2522       2532       2542       2552       2562       2572CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein      2582       2592       2602       2612       2622       2632CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein      2642       2652       2662       2672       2682       2692CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein      2702       2712       2722       2732       2742       2752ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein      2762       2772       2782       2792       2802       2812CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein      2822       2832       2842       2852       2862       2872AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein      2882       2892       2902       2912       2922       2932ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein      2942       2952       2962       2972       2982       2992ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein      3002       3012       3022       3032       3042       3052CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein      3062       3072       3082       3092       3102       3112CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein      3122       3132       3142       3152       3162       3172UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein      3182       3192       3202       3212       3222       3232GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein      3242       3252       3262       3272       3282       3292GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein      3302       3312       3322       3332       3342       3352AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein      3362       3372       3382       3392       3402       3412CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein      3422       3432       3442       3452       3462       3472UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein      3482       3492       3502       3512       3522       3532CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein      3542       3552       3562       3572       3582       3592UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein      3602       3612       3622       3632       3642       3652AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein      3662       3672       3682       3692       3702       3712CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein      3722       3732       3742       3752       3762       3772AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein      3782       3792       3802       3812       3822       3832UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein      3842       3852       3862       3872  3877UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein      3887       3897       3907       3917       3927       3937CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element      3947       3957       3967       3977       3987       3997AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element      4007       4017       4027       4037       4047       4057UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element      4067       4077       4087       4097       4107       4117CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element      4127       4137       4147       4157       4167       4172GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                          FI element      4182       4192       4202       4212       4222       4232AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      4242       4252       4262       4272       4282AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nuleotide sequence (* = stop codon).        10         20         30         40         50 52GGGCGAACUA GUAUUCUUCU GGUCCCCACA GACUCAGAGA GAACCCGCCA CC                           hAg-Kozak        62         72         82         92        102        112AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein       122        132        142        152        162        172AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein       182        192        202        212        222        232AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein       242        252        262        272        282        292AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein       302        312        322        332        342        352AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein       362        372        382        392        402        412AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein       422        432        442        452        462        472AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein       482        492        502        512        522        532CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein       542        552        562        572        582        592UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein       602        612        622        632        642        652GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein       662        672        682        692        702        712UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein       722        732        742        752        762        772UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein       782        792        802        812        822        832CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein       842        852        862        872        882        892GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein       902        912        922        932        942        952GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein       962        972        982        992       1002       1012UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      1022       1032       1042       1052       1062       1072CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      1082       1092       1102       1112       1122       1132GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      1142       1152       1162       1172       1182       1192UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      1202       1212       1222       1232       1242       1252GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      1262       1272       1282       1292       1302       1312GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      1322       1332       1342       1352       1362       1372UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      1382       1392       1402       1412       1422       1432UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      1442       1452       1462       1472       1482       1492CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      1502       1512       1522       1532       1542       1552AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      1562       1572       1582       1592       1602       1612AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      1622       1632       1642       1652       1662       1672CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      1682       1692       1702       1712       1722       1732UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      1742       1752       1762       1772       1782       1792CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      1802       1812       1822       1832       1842       1852ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      1862       1872       1882       1892       1902       1912GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      1922       1932       1942       1952       1962       1972CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      1982       1992       2002       2012       2022       2032AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      2042       2052       2062       2072       2082       2092GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      2102       2112       2122       2132       2142       2152CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      2162       2172       2182       2192       2202       2212GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      2222       2232       2242       2252       2262       2272UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      2282       2292       2302       2312       2322       2332UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      2342       2352       2362       2372       2382       2392ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      2402       2412       2422       2432       2442       2452GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      2462       2472       2482       2492       2502       2512AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein      2522       2532       2542       2552       2562       2572CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein      2582       2592       2602       2612       2622       2632CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein      2642       2652       2662       2672       2682       2692CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein      2702       2712       2722       2732       2742       2752ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein      2762       2772       2782       2792       2802       2812CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein      2822       2832       2842       2852       2862       2872AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein      2882       2892       2902       2912       2922       2932ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein      2942       2952       2962       2972       2982       2992ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein      3002       3012       3022       3032       3042       3052CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein      3062       3072       3082       3092       3102       3112CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein      3122       3132       3142       3152       3162       3172UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein      3182       3192       3202       3212       3222       3232GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein      3242       3252       3262       3272       3282       3292GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein      3302       3312       3322       3332       3342       3352AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein      3362       3372       3382       3392       3402       3412CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein      3422       3432       3442       3452       3462       3472UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein      3482       3492       3502       3512       3522       3532CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein      3542       3552       3562       3572       3582       3592UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein      3602       3612       3622       3632       3642       3652AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein      3662       3672       3682       3692       3702       3712CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein      3722       3732       3742       3752       3762       3772AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein      3782       3792       3802       3812       3822       3832UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein      3842       3852       3862       3872  3877UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein      3887       3897       3907       3917       3927       3937CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element      3947       3957       3967       3977       3987       3997AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element      4007       4017       4027       4037       4047       4057UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element      4067       4077       4087       4097       4107       4117CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element      4127       4137       4147       4157       4167       4172GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                          FI element      4182       4192       4202       4212       4222       4232AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      4242       4252       4262       4272       4282AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40         50 52GGGCGAACUA GUAUUCUUCU GGUCCCCACA GACUCAGAGA GAACCCGCCA CC                           hAg-Kozak        62         72         82         92        102        112AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGGU GAGAUUUCCA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   V  R  F P                          RBD (S protein)       122        132        142        152        162        172AAUAUUACAA AUCUGUGUCC AUUUGGAGAA GUGUUUAAUG CAACAAGAUU UGCAUCUGUG  N  I  T   N  L  C   P  F  G  E   V  F  N   A  T  R   F  A  S  V                          RBD (S protein)       182        192        202        212        222        232UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU  Y  A  W   N  R  K   R  I  S  N   C  V  A   D  Y  S   V  L  Y  N                          RBD (S protein)       242        252        262        272        282        292AGUGCUUCUU UUUCCACAUU UAAAUGUUAU GGAGUGUCUC CAACAAAAUU AAAUGAUUUA  S  A  S   F  S  T   F  K  C  Y   G  V  S   P  T  K   L  N  D  L                          RBD (S protein)       302        312        322        332        342        352UGUUUUACAA AUGUGUAUGC UGAUUCUUUU GUGAUCAGAG GUGAUGAAGU GAGACAGAUU  C  F  T   N  V  Y   A  D  S  F   V  I  R   G  D  E   V  R  Q  I                          RBD (S protein)       362        372        382        392        402        412GCCCCCGGAC AGACAGGAAA AAUUGCUGAU UACAAUUACA AACUGCCUGA UGAUUUUACA  A  P  G   Q  T  G   K  I  A  D   Y  N  Y   K  L  P   D  D  F  T                          RBD (S protein)       422        432        442        452        462        472GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU UUAGAUUCUA AAGUGGGAGG AAAUUACAAU  G  C  V   I  A  W   N  S  N  N   L  D  S   K  V  G   G  N  Y  N                          RBD (S protein)       482        492        502        512        522        532UAUCUGUACA GACUGUUUAG AAAAUCAAAU CUGAAACCUU UUGAAAGAGA UAUUUCAACA  Y  L  Y   R  L  F   R  K  S  N   L  K  P   F  E  R   D  I  S  T                          RBD (S protein)       542        552        562        572        582        592GAAAUUUAUC AGGCUGGAUC AACACCUUGU AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU  E  I  Y   Q  A  G   S  T  P  C   N  G  V   E  G  F   N  C  Y  F                          RBD (S protein)       602        612        622        632        642        652CCAUUACAGA GCUAUGGAUU UCAGCCAACC AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG  P  L  Q   S  Y  G   E  Q  P  T   N  G  V   G  Y  Q   P  Y  R  V                          RBD (S protein)       662        672        682        692        702        706GUGGUGCUGU CUUUUGAACU GCUGCAUGCA CCUGCAACAG UGUGUGGACC UAAA  V  V  L   S  F  E   L  L  H  A   P  A  T   V  C  G   P  K                          RBD (S protein)       716        726        733 GGCUCCCCCG GCUCCGGCUC CGGAUCU  G  S  P   G  S  G   S  G  S                           GS linker       743        753        763        773        783        793GGUUAUAUUC CUGAAGCUCC AAGAGAUGGG CAAGCUUACG UUCGUAAAGA UGGCGAAUGG  G  Y  I   P  E  A   P  R  D  G   Q  A  Y   V  R  K   D  G  E  W                          fibritin       803        813        823        833        843        853GUAUUACUUU CUACCUUUUU AGGCCGGUCC CUGGAGGUGC UGUUCCAGGG CCCCGGCUGA  V  L  L   S  T  F   L  G  R  S   L  E  V   L  F  Q   G  P  G  *                          fibritin 856 UGA   * fibritin       866        876        886        896        906        916CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                           FI element       926        936        946        956        966        976AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                           FI element       986        996       1006       1016       1026       1036UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                           FI element      1046       1056       1066       1076       1086       1096CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                           FI element      1106       1116       1126       1136       1146       1151GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                           FI element      1161       1171       1181       1191       1201       1211AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      1221       1231       1241       1251       1261AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)Nucleoside Modified Messenger RNA (modRNA)

The active principle of the nucleoside modified messenger RNA (modRNA)drug substance is as well a single-stranded mRNA that is translated uponentering a cell. In addition to the sequence encoding the coronavirusvaccine antigen (i.e. open reading frame), each modRNA contains commonstructural elements optimized for maximal efficacy of the RNA as theuRNA (5′-cap, 5′-UTR, 3′-UTR, poly(A)-tail). Compared to the uRNA,modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred5′ cap structure is m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG. The preferred5′-UTR and 3′-UTR comprise the nucleotide sequence of SEQ ID NO: 12 andthe nucleotide sequence of SEQ ID NO: 13, respectively. The preferredpoly(A)-tail comprises the sequence of SEQ ID NO: 14. An additionalpurification step is applied for modRNA to reduce dsRNA contaminantsgenerated during the in vitro transcription reaction.

Different embodiment of this platform are as follows:

BNT162b2; RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7) Structure m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)- hAg-Kozak-S1S2-PP-FI-A30L70 Encodedantigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2full-length protein, sequence variant) BNT162b2; RBP020.2 (SEQ ID NO:20; SEQ ID NO: 7) Structure m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant)BNT162b1; RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5) Structure m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)- hAg-Kozak-RBD-GS-Fibritin-FI-A30L70Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2(partial sequence, Receptor Binding Domain (RBD) of S1S2 protein fusedto fibritin)

FIG. 20 schematizes the general structure of the antigen-encoding RNAs.

Nucleotide Sequence of RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in bold letters.In addition, the sequence of the translated protein is shown in italic letters below thecoding nucleotide sequence (* = stop codon).        10         20         30         40         50  53AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACC                         hAg-Kozak        63         73         83         93        103        113AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein       123        133        143        153        163        173AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein       183        193        203        213        223        233AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein       243        253        263        273        283        293AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein       303        313        323        333        343        353AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein       363        373        383        393        403        413AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein       423        433        443        453        463        473AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein       483        493        503        513        523        533CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein       543        553        563        573        583        593UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein       603        613        623        633        643        653GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein       663        673        683        693        703        713UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein       723        733        743        753        763        773UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein       783        793        803        813        823        833CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein       843        853        863        873        883        893GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein       93        913        923        933        943        953GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein       963        973        983        993       1003       1013UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      1023       1033       1043       1053       1063       1073CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      1083       1093       1103       1113       1123       1133GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      1143       1153       1163       1173       1183       1193UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      1203       1213       1223       1233       1243       1253GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      1262       1272       1282       1292       1302       1312GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      1323       1333       1343       1353       1363       1373UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      1383       1393       1403       1413       1423       1433UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      1443       1453       1463       1473       1483       1493CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      1503       1513       1523       1533       1543       1553AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      1563       1573       1583       1593       1603       1613AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      1623       1633       1643       1653       1663       1673CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      1683       1693       1703       1713       1723       1733UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      1743       1753       1763       1773       1783       1793CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      1803       1813       1823       1833       1843       1853ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      1863       1873       1883       1893       1903       1913GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      1923       1933       1943       1953       1963       1973CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      1983       1993       2003       2013       2023       2033AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      2043       2053       2063       2073       2083       2093GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      2103       2113       2123       2133       2143       2153CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      2163       2173       2183       2193       2203       2213GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      2223       2233       2243       2253       2263       2273UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      2283       2293       2303       2313       2323       2333UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      2343       2353       2363       2373       2383       2393ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      2403       2413       2423       2433       2443       2453GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      2463       2473       2483       2493       2503       2513AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein      2523       2533       2543       2553       2563       2573CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein      2583       2593       2603       2613       2623       2633CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein      2643       2653       2663       2673       2683       2693CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein      2703       2713       2723       2733       2743       2753ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein      2763       2773       2783       2793       2803       2813CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein      2823       2833       2843       2853       2863       2873AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein      2883       2893       2903       2913       2923       2933ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein      2943       2953       2963       2973       2983       2993ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein      3003       3013       3023       3033       3043       3053CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein      3063       3073       3083       3093       3103       3113CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein      3123       3133       3143       3153       3163       3173UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein      3183       3193       3203       3213       3223       3233GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein      3243       3253       3263       3273       3283       3293GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein      3303       3313       3323       3333       3343       3353AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein      3363       3373       3383       3393       3403       3413CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein      3423       3433       3443       3453       3463       3473UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein      3483       3493       3503       3513       3523       3533CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein      3543       3553       3563       3573       3583       3593UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein      3603       3613       3623       3633       3643       3653AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein      3663       3673       3683       3693       3703       3713CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein      3723       3733       3743       3753       3763       3773AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein      3783       3793       3803       3813       3823       3833UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein      3843       3853       3863       3873  3878UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein      3888       3898       3908       3918       3928       3938CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                           FI element      3948       3958       3968       3978       3988       3998AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                           FI element      4088       4018       4028       4038       4048       4058UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                           FI element      4068       4078       4088       4098       4108       4118CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                           FI element      4128       4138       4148       4158       4168  4173GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                           FI element      4183       4193       4203       4213       4223       4233AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      4243       4253       4263       4273       4283AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in bold letters.In addition, the sequence of the translated protein is shown in italic letters below thecoding nucleotide sequence (* = stop codon).        10         20         30         40         50  53AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACC                         hAg-Kozak        63         73         83         93        103        113AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein       123        133        143        153        163        173AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein       183        193        203        213        223        233AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein       243        253        263        273        283        293AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein       303        313        323        333        343        353AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein       363        373        383        393        403        413AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein       423        433        443        453        463        473AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein       483        493        503        513        523        533CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein       543        553        563        573        583        593UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein       603        613        623        633        643        653GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein       663        673        683        693        703        713UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein       723        733        743        753        763        773UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein       783        793        803        813        823        833CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein       843        853        863        873        883        893GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein       93        913        923        933        943        953GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein       963        973        983        993       1003       1013UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      1023       1033       1043       1053       1063       1073CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      1083       1093       1103       1113       1123       1133GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      1143       1153       1163       1173       1183       1193UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      1203       1213       1223       1233       1243       1253GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      1262       1272       1282       1292       1302       1312GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      1323       1333       1343       1353       1363       1373UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      1383       1393       1403       1413       1423       1433UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      1443       1453       1463       1473       1483       1493CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      1503       1513       1523       1533       1543       1553AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      1563       1573       1583       1593       1603       1613AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      1623       1633       1643       1653       1663       1673CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      1683       1693       1703       1713       1723       1733UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      1743       1753       1763       1773       1783       1793CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      1803       1813       1823       1833       1843       1853ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      1863       1873       1883       1893       1903       1913GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      1923       1933       1943       1953       1963       1973CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      1983       1993       2003       2013       2023       2033AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      2043       2053       2063       2073       2083       2093GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      2103       2113       2123       2133       2143       2153CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      2163       2173       2183       2193       2203       2213GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      2223       2233       2243       2253       2263       2273UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      2283       2293       2303       2313       2323       2333UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      2343       2353       2363       2373       2383       2393ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      2403       2413       2423       2433       2443       2453GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      2463       2473       2483       2493       2503       2513AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein      2523       2533       2543       2553       2563       2573CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein      2583       2593       2603       2613       2623       2633CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein      2643       2653       2663       2673       2683       2693CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein      2703       2713       2723       2733       2743       2753ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein      2763       2773       2783       2793       2803       2813CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein      2823       2833       2843       2853       2863       2873AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein      2883       2893       2903       2913       2923       2933ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein      2943       2953       2963       2973       2983       2993ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein      3003       3013       3023       3033       3043       3053CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein      3063       3073       3083       3093       3103       3113CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein      3123       3133       3143       3153       3163       3173UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein      3183       3193       3203       3213       3223       3233GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein      3243       3253       3263       3273       3283       3293GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein      3303       3313       3323       3333       3343       3353AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein      3363       3373       3383       3393       3403       3413CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein      3423       3433       3443       3453       3463       3473UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein      3483       3493       3503       3513       3523       3533CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein      3543       3553       3563       3573       3583       3593UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein      3603       3613       3623       3633       3643       3653AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein      3663       3673       3683       3693       3703       3713CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein      3723       3733       3743       3753       3763       3773AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein      3783       3793       3803       3813       3823       3833UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein      3843       3853       3863       3873  3878UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein      3888       3898       3908       3918       3928       3938CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                           FI element      3948       3958       3968       3978       3988       3998AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                           FI element      4088       4018       4028       4038       4048       4058UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                           FI element      4068       4078       4088       4098       4108       4118CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                           FI element      4128       4138       4148       4158       4168  4173GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                           FI element      4183       4193       4203       4213       4223       4233AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      4243       4253       4263       4273       4283AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5)Nucleotide sequence is shown with individual sequence elements as indicated in bold letters.In addition, the sequence of the translated protein is shown in italic letters below thecoding nucleotide sequence (* = stop codon).        10         20         30         40         50 53GGGCGAACUA GUAUUCUUCU GGUCCCCACA GACUCAGAGA GAACCCGCCA CC                           hAg-Kozak        63         73         83         93        103        1123AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGGU GAGAUUUCCA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   V  R  F P                          RBD (S protein)       123        133        143        153        163        173AAUAUUACAA AUCUGUGUCC AUUUGGAGAA GUGUUUAAUG CAACAAGAUU UGCAUCUGUG  N  I  T   N  L  C   P  F  G  E   V  F  N   A  T  R   F  A  S  V                          RBD (S protein)       183        193        203        213        223        233UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU  Y  A  W   N  R  K   R  I  S  N   C  V  A   D  Y  S   V  L  Y  N                          RBD (S protein)       243        253        263        273        283        293AGUGCUUCUU UUUCCACAUU UAAAUGUUAU GGAGUGUCUC CAACAAAAUU AAAUGAUUUA  S  A  S   F  S  T   F  K  C  Y   G  V  S   P  T  K   L  N  D  L                          RBD (S protein)       303        313        323        333        343        353UGUUUUACAA AUGUGUAUGC UGAUUCUUUU GUGAUCAGAG GUGAUGAAGU GAGACAGAUU  C  F  T   N  V  Y   A  D  S  F   V  I  R   G  D  E   V  R  Q  I                          RBD (S protein)       363        373        383        393        403        413GCCCCCGGAC AGACAGGAAA AAUUGCUGAU UACAAUUACA AACUGCCUGA UGAUUUUACA  A  P  G   Q  T  G   K  I  A  D   Y  N  Y   K  L  P   D  D  F  T                          RBD (S protein)       423        433        443        453        463        473GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU UUAGAUUCUA AAGUGGGAGG AAAUUACAAU  G  C  V   I  A  W   N  S  N  N   L  D  S   K  V  G   G  N  Y  N                          RBD (S protein)       483        43        503        513        523        533UAUCUGUACA GACUGUUUAG AAAAUCAAAU CUGAAACCUU UUGAAAGAGA UAUUUCAACA  Y  L  Y   R  L  F   R  K  S  N   L  K  P   F  E  R   D  I  S  T                          RBD (S protein)       543        553        563        573        583        593GAAAUUUAUC AGGCUGGAUC AACACCUUGU AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU  E  I  Y   Q  A  G   S  T  P  C   N  G  V   E  G  F   N  C  Y  F                          RBD (S protein)       603        613        623        633        643        653CCAUUACAGA GCUAUGGAUU UCAGCCAACC AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG  P  L  Q   S  Y  G   E  Q  P  T   N  G  V   G  Y  Q   P  Y  R  V                          RBD (S protein)       663        673        683        693        703   707GUGGUGCUGU CUUUUGAACU GCUGCAUGCA CCUGCAACAG UGUGUGGACC UAAA  V  V  L   S  F  E   L  L  H  A   P  A  T   V  C  G   P  K                          RBD (S protein)        717        727     734GGCUCCCCCG GCUCCGGCUC CGGAUCU   G  S  P   G  S  G   S  G  S                          GS linker       744        754        764        774        784        794GGUUAUAUUC CUGAAGCUCC AAGAGAUGGG CAAGCUUACG UUCGUAAAGA UGGCGAAUGG  G  Y  I   P  E  A   P  R  D  G   Q  A  Y   V  R  K   D  G  E  W                          fibritin       804        814        824        834        844        854GUAUUACUUU CUACCUUUUU AGGCCGGUCC CUGGAGGUGC UGUUCCAGGG CCCCGGCUGA  V  L  L   S  T  F   L  G  R  S   L  E  V   L  F  Q   G  P  G  *                          fibritin 857 UGA   * fibritin       867        877        887        897        907        917CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                           FI element       927        937        947        957        967        977AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                           FI element       987        997       1007       1017       1027       1037UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                           FI element      1047       1057       1067       1077       1087       1097CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                           FI element      1107       1117       1127       1137       1147  1152GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                           FI element      1162       1172       1182      1192        1202       1212AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      1222       1232       1242       1252       1262AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Further embodiments of the nucleoside modified messenger RNA (modRNA)platform are as follows:

BNT162b3c (SEQ ID NO: 29; SEQ ID NO: 30) Structure m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A3OL70Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence,Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused to TransmembraneDomain (TM) of S1S2 protein); intrinsic S1S2 protein secretory signal peptide (aa 1-19) atthe N-terminus of the antigen sequenceagaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc acc aug  56                                                           Met                                                           1uuu gug uuu cuu gug cug cug ccu cuu gug ucu ucu cag ugu gug aau  104Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val Asn            5                   10                  15uug aca gug aga uuu cca aau auu aca aau cug ugu cca uuu gga gaa  152Leu Thr Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu        20                  25                  30gug uuu aau gca aca aga uuu gca ucu gug uau gca ugg aau aga aaa  200Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys    35                  40                  45aga auu ucu aau ugu gug gcu gau uau ucu gug cug uau aau agu gcu  248Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala50                  55                  60                  65ucu uuu ucc aca uuu aaa ugu uau gga gug ucu cca aca aaa uua aau  296Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn                70                  75                  80gau uua ugu uuu aca aau gug uau gcu gau ucu uuu gug auc aga ggu  344Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly            85                  90                   95gau gaa gug aga cag auu gcc ccc gga cag aca gga aaa auu gcu gau  392Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp        100                 105                 110uac aau uac aaa cug ccu gau gau uuu aca gga ugu gug auu gcu ugg  440Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp    115                 120                 125aau ucu aau aau uua gau ucu aaa gug gga gga aau uac aau uau cug  488Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu130                 135                 140                 145uac aga cug uuu aga aaa uca aau cug aaa ccu uuu gaa aga gau auu  536Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile                150                 155                 160uca aca gaa auu uau cag gcu gga uca aca ccu ugu aau gga gug gaa  584Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu            165                 170                 175gga uuu aau ugu uau uuu cca uua cag agc uau gga uuu cag cca acc  632Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr        180                 185                 190aau ggu gug gga uau cag cca uau aga gug gug gug cug ucu uuu gaa  680Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu    195                 200                 205cug cug cau gca ccu gca aca gug ugu gga ccu aaa ggc ucc ccc ggc  728Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Gly Ser Pro Gly210                 215                 220                 225ucc ggc ucc gga ucu ggu uau auu ccu gaa gcu cca aga gau ggg caa  776Ser Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln                230                 235                 240gcu uac guu cgu aaa gau ggc gaa ugg gua uua cuu ucu acc uuu uua  824Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu            245                 250                 255gga agc ggc agc gga ucu gaa cag uac auu aaa ugg ccu ugg uac auu  872Gly Ser Gly Ser Gly Ser Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile        260                 265                 270ugg cuu gga uuu auu gca gga uua auu gca auu gug aug gug aca auu  920Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile    275                 280                 285aug uua ugu ugu aug aca uca ugu ugu ucu ugu uua aaa gga ugu ugu  968Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys290                 295                 300                 305ucu ugu gga agc ugu ugu uga ugacucgagc ugguacugca ugcacgcaau 1019Ser Cys Gly Ser Cys Cys                     310gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc cccgaccucg ggucccaggu 1079augcucccac cuccaccugc cccacucacc accucugcua guuccagaca ccucccaagc 1139acgcagcaau gcagcucaaa acgcuuagcc uagccacacc cccacgggaa acagcaguga 1199uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac uaaccccagg guuggucaau 1259uucgugccag ccacacccug gagcuagcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagc 1319auaugacuaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1379aaaaaaaaaa aaaaaaaa 1397

BNT162b3d (SEQ ID NO: 31; SEQ ID NO: 32) Structure m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence,Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fusedto Transmembrane Domain (TM) of S1S2 protein); immunoglobulin secretorysignal peptide (aa 1-22) at the N-terminus of the antigen sequenceagaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc acc aug   56                                                           Met                                                           1gau ugg auu ugg aga auc cug uuc cuc gug gga gcc gcu aca gga gcc  104Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly Ala            5                   10                  15cac ucc cag aug cag gug aga uuu cca aau auu aca aau cug ugu cca  152His Ser Gln Met Gln Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro        20                  25                  30uuu gga gaa gug uuu aau gca aca aga uuu gca ucu gug uau gca ugg  200Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp    35                  40                  45aau aga aaa aga auu ucu aau ugu gug gcu gau uau ucu gug cug uau  248Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr50                  55                  60                  65aau agu gcu ucu uuu ucc aca uuu aaa ugu uau gga gug ucu cca aca  296Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr                70                  75                  80aaa uua aau gau uua ugu uuu aca aau gug uau gcu gau ucu uuu gug  344Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val            85                  90                   95auc aga ggu gau gaa gug aga cag auu gcc ccc gga cag aca gga aaa  392Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys        100                 105                 110auu gcu gau uac aau uac aaa cug ccu gau gau uuu aca gga ugu gug  440Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val    115                 120                 125auu gcu ugg aau ucu aau aau uua gau ucu aaa gug gga gga aau uac  488Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr130                 135                 140                 145aau uau cug uac aga cug uuu aga aaa uca aau cug aaa ccu uuu gaa  536Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu                150                 155                 160aga gau auu uca aca gaa auu uau cag gcu gga uca aca ccu ugu aau  584Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn            165                 170                 175gga gug gaa gga uuu aau ugu uau uuu cca uua cag agc uau gga uuu  632Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe        180                 185                 190cag cca acc aau ggu gug gga uau cag cca uau aga gug gug gug cug  680Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu    195                 200                 205ucu uuu gaa cug cug cau gca ccu gca aca gug ugu gga ccu aaa ggc  728Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Gly210                 215                 220                 225ucc ccc ggc ucc ggc ucc gga ucu ggu uau auu ccu gaa gcu cca aga  776Ser Pro Gly Ser Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg                230                 235                 240gau ggg caa gcu uac guu cgu aaa gau ggc gaa ugg gua uua cuu ucu  824Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser            245                 250                 255acc uuu uua gga agc ggc agc gga ucu gaa cag uac auu aaa ugg ccu  872Thr Phe Leu Gly Ser Gly Ser Gly Ser Glu Gln Tyr Ile Lys Trp Pro        260                 265                 270ugg uac auu ugg cuu gga uuu auu gca gga uua auu gca auu gug aug  920Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met    275                 280                 285gug aca auu aug uua ugu ugu aug aca uca ugu ugu ucu ugu uua aaa  968Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys290                 295                 300                 305gga ugu ugu ucu ugu gga agc ugu ugu uga ugacucgagc ugguacugca 1018Gly Cys Cys Ser Cys Gly Ser Cys Cys                 310ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc cccgaccucg 1078ggucccaggu augcucccac cuccaccugc cccacucacc accucugcua guuccagaca 1138ccucccaagc acgcagcaau gcagcucaaa acgcuuagcc uagccacacc cccacgggaa 1198acagcaguga uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac uaaccccagg 1258guuggucaau uucgugccag ccacacccug gagcuagcaa aaaaaaaaaa aaaaaaaaaa 1318aaaaaaaagc auaugacuaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1378aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1406Self-Amplifying RNA (saRNA)

The active principle of the self-amplifying mRNA (saRNA) drug substanceis a single-stranded RNA, which self-amplifies upon entering a cell, andthe coronavirus vaccine antigen is translated thereafter. In contrast touRNA and modRNA that preferably code for a single protein, the codingregion of saRNA contains two open reading frames (ORFs). The 5′-ORFencodes the RNA-dependent RNA polymerase such as Venezuelan equineencephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). Thereplicase ORF is followed 3′ by a subgenomic promoter and a second ORFencoding the antigen. Furthermore, saRNA UTRs contain 5′ and 3′conserved sequence elements (CSEs) required for self-amplification. ThesaRNA contains common structural elements optimized for maximal efficacyof the RNA as the uRNA (5′-cap, 5′-UTR, 3′-UTR, poly(A)-tail). The saRNApreferably contains uridine. The preferred 5′ cap structure isbeta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG).

Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle.However, the saRNA does not encode for alphaviral structural proteinsthat are required for genome packaging or cell entry, thereforegeneration of replication competent viral particles is very unlikely tonot possible. Replication does not involve any intermediate steps thatgenerate DNA. The use/uptake of saRNA therefore poses no risk of genomicintegration or other permanent genetic modification within the targetcell. Furthermore, the saRNA itself prevents its persistent replicationby effectively activating innate immune response via recognition ofdsRNA intermediates.

Different embodiment of this platform are as follows:

RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spikeprotein (S protein) of the SARS-CoV-2 (S1S2 full-length protein,sequence variant) RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spikeprotein (S protein) of the SARS-CoV-2 (S1S2 full-length protein,sequence variant) BNT162C1; RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5)Structure beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-FI-A30L70 Encodedantigen Viral spike protein (S protein) of the SARS-CoV-2 (partialsequence, Receptor Binding Domain (RBD) of S1S2 protein) RBS004.4 (SEQID NO: 27; SEQ ID NO: 28) Structurebeta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-TM-FI-A30L70 Encoded antigenViral spike protein (S protein) of the SARS-CoV-2 (partial sequence,Receptor Binding Domain (RBD) of S1S2 protein)

FIG. 21 schematizes the general structure of the antigen-encoding RNAs.

Nucleotide Sequence of RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40    45GAUGGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA                           5′UTR        55         65         75         85         95        105AUGGAGAAAG UUCACGUUGA CAUCGAGGAA GACAGCCCAU UCCUCAGAGC UUUGCAGCGG  M  E  K   V  H  V   D  I  E  E   D  S  P   F  L  R   A  L  Q  R                            nsp1       115        125        135        145        155        165AGCUUCCCGC AGUUUGAGGU AGAAGCCAAG CAGGUCACUG AUAAUGACCA UGCUAAUGCC  S  F  P   Q  F  E   V  E  A  K   Q  V  T   D  N  D   H  A  N  A                            nsp1       175        185        195        205        215        225AGAGCGUUUU CGCAUCUGGC UUCAAAACUG AUCGAAACGG AGGUGGACCC AUCCGACACG  R  A  F   S  H  L   A  S  K  L  I  E  T    E  V  D   P  S  D  T                            nsp1       235        245        255        265        275        285AUCCUUGACA UUGGAAGUGC GCCCGCCCGC AGAAUGUAUU CUAAGCACAA GUAUCAUUGU  I  L  D   I  G  S   A  P  A  R  R  M  Y    S  K  H   K  Y  H  C                            nsp1       295        305        315        325        335        345AUCUGUCCGA UGAGAUGUGC GGAAGAUCCG GACAGAUUGU AUAAGUAUGC AACUAAGCUG I  C  P    M  R  C   A  E  D  P   D  R  L   Y  K  Y   A  T  K  L                            nsp1       355        365        375        385        395        405AAGAAAAACU GUAAGGAAAU AACUGAUAAG GAAUUGGACA AGAAAAUGAA GGAGCUCGCC  K  K  N   C  K  E   I  T  D  K   E  L  D   K  K  M   K  E  L  A                            nsp1       415        425        435        445        455        465GCCGUCAUGA GCGACCCUGA CCUGGAAACU GAGACUAUGU GCCUCCACGA CGACGAGUCG  A  V  M   S  D  P   D  L  E  T   E  T  M   C  L  H   D  D  E  S                            nsp1       475        485        495        505        515        525UGUCGCUACG AAGGGCAAGU CGCUGUUUAC CAGGAUGUAU ACGCGGUUGA CGGACCGACA  C  R  Y   E  G  Q   V  A  V  Y   Q  D  V   Y  A  V   D  G  P  T                            nsp1       535        545        555        565        575        585AGUCUCUAUC ACCAAGCCAA UAAGGGAGUU AGAGUCGCCU ACUGGAUAGG CUUUGACACC  S  L  Y   H  Q  A   N  K  G  V   R  V  A   Y  W  I   G  F  D  T                            nsp1       595        605        615        625        635        645ACCCCUUUUA UGUUUAAGAA CUUGGCUGGA GCAUAUCCAU CAUACUCUAC CAACUGGGCC  T  P  F   M  F  K   N  L  A  G   A  Y  P   S  Y  S   T  N  W  A                            nsp1       655        665        675        685        695        705GACGAAACCG UGUUAACGGC UCGUAACAUA GGCCUAUGCA GCUCUGACGU UAUGGAGCGG  D  E  T   V  L  T   A  R  N  I   G  L  C   S  S  D   V  M  E  R                            nsp1       715        725        735        745        755        765UCACGUAGAG GGAUGUCCAU UCUUAGAAAG AAGUAUUUGA AACCAUCCAA CAAUGUUCUA  S  R  R   G  M  S   I  L  R  K   K  Y  L   K  P  S   N  N  V  L                            nsp1       775        785        795        805        815        825UUCUCUGUUG GCUCGACCAU CUACCACGAA AAGAGGGACU UACUGAGGAG CUGGCACCUG  F  S  V   G  S  T   I  Y  H  E   K  R  D   L  L  R   S  W  H  L                            nsp1       835        845        855        865        875        885CCGUCUGUAU UUCACUUACG UGGCAAGCAA AAUUACACAU GUCGGUGUGA GACUAUAGUU  P  S  V   F  H  L   R  G  K  Q   N  Y  T   C  R  C   E  T  I  V                            nsp1       895        905        915        925        935        945AGUUGCGACG GGUACGUCGU UAAAAGAAUA GCUAUCAGUC CAGGCCUGUA UGGGAAGCCU  S  C  D   G  Y  V   V  K  R  I   A  I  S   P  G  L   Y  G  K  P                            nsp1       955        965        975        985        995       1005UCAGGCUAUG CUGCUACGAU GCACCGCGAG GGAUUCUUGU GCUGCAAAGU GACAGACACA  S  G  Y   A  A  T   M  H  R  E   G  E  L   C  C  K   V  T  D  T                            nsp1      1015       1025       1035       1045       1055       1065UUGAACGGGG AGAGGGUCUC UUUUCCCGUG UGCACGUAUG UGCCAGCUAC AUUGUGUGAC  L  N  G   E  R  V   S  F  P  V   C  T  Y   V  P  A   T  L  C  D                            nsp1      1075       1085       1095       1105       1115       1125CAAAUGACUG GCAUACUGGC AACAGAUGUC AGUGCGGACG ACGCGCAAAA ACUGCUGGUU  Q  M  T   G  I  L   A  T  D  V   S  A  D   D  A  Q   K  L  L  V                            nsp1      1135       1145       1155       1165       1175       1185GGGCUCAACC AGCGUAUAGU CGUCAACGGU CGCACCCAGA GAAACACCAA UACCAUGAAA  G  L  N   Q  R  I   V  V  N  G   R  T  Q   R  N  T   N  T  M  K                            nsp1      1198       1205       1215       1225       1235       1245AAUUACCUUU UGCCCGUAGU GGCCCAGGCA UUUGCUAGGU GGGCAAAGGA AUAUAAGGAA  N  Y  L   L  P  V   V  A  Q  A   F  A  R   W  A  K   E  Y  K  E                            nsp1      1255       1265       1275       1285       1295       1305GAUCAAGAAG AUGAAAGGCC ACUAGGACUA CGAGAUAGAC AGUUAGUCAU GGGGUGUUGU  D  Q  E   D  E  R   P  L  G  L   R  D  R   Q  L  V   M  G  C  C                            nsp1      1315       1325       1335       1345       1355       1365UGGGCUUUUA GAAGGCACAA GAUAACAUCU AUUUAUAAGC GCCCGGAUAC CCAAACCAUC  W  A  F   R  R  H   K  I  T  S   I  Y  K   R  P  D  T  Q  T  I                            nsp1      1375       1385       1395       1405       1415       1425AUCAAAGUGA ACAGCGAUUU CCACUCAUUC GUGCUGCCCA GGAUAGGCAG UAACACAUUG  I  K  V   N  S  D   F  H  S  F   V  L  P   R  I  G   S  N  T  L                            nsp1      1435       1445       1455       1465       1475       1485GAGAUCGGGC UGAGAACAAG AAUCAGGAAA AUGUUAGAGG AGCACAAGGA GCCGUCACCU  E  I  G   L  R  T   R  I  R  K   M  L  E   E  H  K   E  P  S  P                            nsp1      1495       1505       1515       1525       1535       1545CUCAUUACCG CCGAGGACGU ACAAGAAGCU AAGUGCGCAG CCGAUGAGGC UAAGGAGGUG  L  I  T   A  E  D   V  Q  E  A   K  C  A   A  D  E   A  K  E  V                            nsp1      1555       1565       1575       1585       1595       1605CGUGAAGCCG AGGAGUUGCG CGCAGCUCUA CCACCUUUGG CAGCUGAUGU UGAGGAGCCC  R  E  A   E  E  L   R  A  A  L   P  P  L   A  A  D   V  E  E  P                            nsp1      1615       1625       1635       1645       1650ACUCUGGAAG CCGAUGUCGA CUUGAUGUUA CAAGAGGCUG GGGCC  T  L  E   A  D  V   D  L  M  L   Q  E  A   G  A                            nsp1      1660       1670       1680       1690       1700       1710GGCUCAGUGG AGACACCUCG UGGCUUGAUA AAGGUUACCA GCUACGCUGG CGAGGACAAG  G  S  V   E  T  P   R  G  L  I   K  V  T   S  Y  A   G  E  D  K                            nsp2      1720       1730       1740       1750       1760       1770AUCGGCUCUU ACGCUGUGCU UUCUCCGCAG GCUGUACUCA AGAGUGAAAA AUUAUCUUGC  I  G  S   Y  A  V   L  S  P  Q   A  V  L   K  S  E   K  L  S  C                            nsp2      1780       1790       1800       1810       1820       1830AUCCACCCUC UCGCUGAACA AGUCAUAGUG AUAACACACU CUGGCCGAAA AGGGCGUUAU  I  H  P   L  A  E   Q  V  I  V   I  T  H   S  G  R   K  G  R  Y                            nsp2      1840       1850       1860       1870       1880       1890GCCGUGGAAC CAUACCAUGG UAAAGUAGUG GUGCCAGAGG GACAUGCAAU ACCCGUCCAG  A  V  E   P  Y  H   G  K  V  V   V  P  E   G  H  A   I  P  V  Q                            nsp2      1900       1910       1920       1930       1940       1950GACUUUCAAG CUCUGAGUGA AAGUGCCACC AUUGUGUACA ACGAACGUGA GUUCGUAAAC  D  F  Q   A  L  S   E  S  A  T   I  V  Y   N  E  R   E  F  V  N                            nsp2      1960       1970       1980       1990       2000       2010AGGUACCUGC ACCAUAUUGC CACACAUGGA GGAGCGCUGA ACACUGAUGA AGAAUAUUAC  R  Y  L   H  H  I   A  T  H  G   G  A  L   N  T  D   E  E  Y  Y                            nsp2      2020       2030       2040       2050       2060       2070AAAACUGUCA AGCCCAGCGA GCACGACGGC GAAUACCUGU ACGACAUCGA CAGGAAACAG  K  T  V   K  P  S   E  H  D  G   E  Y  L   Y  D  I   D  R  K  Q                            nsp2      2080       2090       2100       2110       2120       2130UGCGUCAAGA AAGAGCUAGU CACUGGGCUA GGGCUCACAG GCGAGCUGGU CGAUCCUCCC  C  V  K   K  E  L   V  T  G  L   G  L  T   G  E  L   V  D  P  P                            nsp2      2140       2150       2160       2170       2180       2190UUCCAUGAAU UCGCCUACGA GAGUCUGAGA ACACGACCAG CCGCUCCUUA CCAAGUACCA  F  H  E   F  A  Y   E  S  L  R   T  R  P   A  A  P   Y  Q  V  P                            nsp2      2200       2210       2220       2230       2240       2250ACCAUAGGGG UGUAUGGCGU GCCAGGAUCA GGCAAGUCUG GCAUCAUUAA AAGCGCAGUC  T  I  G   V  Y  G   V  P  G  S   G  K  S   G  I  I   K  S  A  V                            nsp2      2260       2270       2280       2290       2300       2310ACCAAAAAAG AUCUAGUGGU GAGCGCCAAG AAAGAAAACU GUGCAGAAAU UAUAAGGGAC  T  K  K   D  L  V   V  S  A  K   K  E  N   C  A  E   I  I  R  D                            nsp2      2320       2330       2340       2350       2360       2370GUCAAGAAAA UGAAAGGGCU GGACGUCAAU GCCAGAACUG UGGACUCAGU GCUCUUGAAU  V  K  K   M  K  G   L  D  V  N   A  R  T   V  D  S   V  L  L  N                            nsp2      2380       2390       2400       2410       2420       2430GGAUGCAAAC ACCCCGUAGA GACCCUGUAU AUUGACGAGG CUUUUGCUUG UCAUGCAGGU  G  C  K   H  P  V   E  T  L  Y   I  D  E   A  F  A   C  H  A  G                            nsp2      2440       2450       2460       2470       2480       2490ACUCUCAGAG CGCUCAUAGC CAUUAUAAGA CCUAAAAAGG CAGUGCUCUG CGGAGAUCCC  T  L  R   A  L  I   A  I  I  R   P  K  K   A  V  L   C  G  D  P                            nsp2      2500       2510       2520       2530       2540       2550AAACAGUGCG GUUUUUUUAA CAUGAUGUGC CUGAAAGUGC AUUUUAACCA CGAGAUUUGC  K  Q  C   G  E  E   N  M  M  C   L  K  V   H  F  N   H  E  I  C                            nsp2      2560       2570       2580       2590       2600       2610ACACAAGUCU UCCACAAAAG CAUCUCUCGC CGUUGCACUA AAUCUGUGAC UUCGGUCGUC  T  Q  V   F  H  K   S  I  S  R   R  C  T   K  S  V   T  S  V  V                            nsp2      2620       2630       2640       2650       2660       2670UCAACCUUGU UUUACGACAA AAAAAUGAGA ACGACGAAUC CGAAAGAGAC UAAGAUUGUG  S  T  L   F  Y  D   K  K  M  R   T  T  N   P  K  E   T  K  I  V                            nsp2      2680       2690       2700       2710       2720       2730AUUGACACUA CCGGCAGUAC CAAACCUAAG CAGGACGAUC UCAUUCUCAC UUGUUUCAGA  I  D  T   T  G  S   T  K  P  K   Q  D  D   L  I  L   T  C  F  R                            nsp2      2740       2750       2760       2770       2780       2790GGGUGGGUGA AGCAGUUGCA AAUAGAUUAC AAAGGCAACG AAAUAAUGAC GGCAGCUGCC  G  W  V   K  Q  L   Q  I  D  Y   K  G  N   E  I  M   T  A  A  A                            nsp2      2800       2810       2820       2830       2840       2850UCUCAAGGGC UGACCCGUAA AGGUGUGUAU GCCGUUCGGU ACAAGGUGAA UGAAAAUCCU  S  Q  G   L  T  R   K  G  V  Y   A  V  R   Y  K  V   N  E  N  P                            nsp2      2860       2870       2880       2890       2900       2910CUGUACGCAC CCACCUCAGA ACAUGUGAAC GUCCUACUGA CCCGCACGGA GGACCGCAUC  L  Y  A   P  T  S   E  H  V  N   V  L  L   T  R  T   E  D  R  I                            nsp2      2920       2930       2940       2950       2960       2970GUGUGGAAAA CACUAGCCGG CGACCCAUGG AUAAAAACAC UGACUGCCAA GUACCCUGGG  V  W  K   T  L  A   G  D  P  W   I  K  T   L  T  A   K  Y  P  G                            nsp2      2980       2990       3000       3010       3020       3030AAUUUCACUG CCACGAUAGA GGAGUGGCAA GCAGAGCAUG AUGCCAUCAU GAGGCACAUC  N  F  T   A  T  I   E  E  W  Q   A  E  H   D  A  I   M  R  H  I                            nsp2      3040       3050       3060       3070       3080       3090UUGGAGAGAC CGGACCCUAC CGACGUCUUC CAGAAUAAGG CAAACGUGUG UUGGGCCAAG  L  E  R   P  D  P   T  D  V  F   Q  N  K   A  N  V   C  W  A  K                            nsp2      3100       3110       3120       3130       3140       3150GCUUUAGUGC CGGUGCUGAA GACCGCUGGC AUAGACAUGA CCACUGAACA AUGGAACACU  A  L  V   P  V  L   K  T  A  G   I  D  M   T  T  E   Q  W  N  T                            nsp2      3160       3170       3180       3190       3200       3210GUGGAUUAUU UUGAAACGGA CAAAGCUCAC UCAGCAGAGA UAGUAUUGAA CCAACUAUGC  V  D  Y   F  E  T   D  K  A  H   S  A  E   I  V  L   N  Q  L  C                            nsp2      3220       3230       3240       3250       3260       3270GUGAGGUUCU UUGGACUCGA UCUGGACUCC GGUCUAUUUU CUGCACCCAC UGUUCCGUUA  V  R  F   F  G  L   D  L  D  S   G  L  F   S  A  P   T  V  P  L                            nsp2      3280       3290       3300       3310       3320       3330UCCAUUAGGA AUAAUCACUG GGAUAACUCC CCGUCGCCUA ACAUGUACGG GCUGAAUAAA  S  I  R   N  N  H   W  D  N  S   P  S  P   N  M  Y   G  L  N  K                            nsp2      3340       3350       3360       3370       3380       3390GAAGUGGUCC GUCAGCUCUC UCGCAGGUAC CCACAACUGC CUCGGGCAGU UGCCACUGGU  E  V  V   R  Q  L   S  R  R  Y   P  Q  L   P  R  A   V  A  T  G                            nsp2      3400       3410       3420       3430       3440       3450AGAGUCUAUG ACAUGAACAC UGGUACACUG CGCAAUUAUG AUCCGCGCAU AAACCUAGUA  R  V  Y   D  M  N   T  G  T  L   R  N  Y   D  P  R   I  N  L  V                            nsp2      3460       3470       3480       3490       3500       3510CCUGUAAACA GAAGACUGCC UCAUGCUUUA GUCCUCCACC AUAAUGAACA CCCACAGAGU  P  V  N   R  R  L   P  H  A  L   V  L  H   H  N  E   H  P  Q  S                            nsp2      3520       3530       3540       3550       3560       3570GACUUUUCUU CAUUCGUCAG CAAAUUGAAG GGCAGAACUG UCCUGGUGGU CGGGGAAAAG  D  F  S   S  F  V   S  K  L  K   G  R  T   V  L  V   V  G  E  K                            nsp2      3580       3590       3600       3610       3620       3630UUGUCCGUCC CAGGCAAAAU GGUUGACUGG UUGUCAGACC GGCCUGAGGC UACCUUCAGA  L  S  V   P  G  K   M  V  D  W   L  S  D   R  P  E   A  T  E  R                            nsp2      3640       3650       3660       3670       3680       3690GCUCGGCUGG AUUUAGGCAU CCCAGGUGAU GUGCCCAAAU AUGACAUAAU AUUUGUUAAU  A  R  L   D  L  G   I  P  G  D   V  P  K   Y  D  I   I  F  V  N                            nsp2      3700       3710       3720       3730       3740       3750GUGAGGACCC CAUAUAAAUA CCAUCACUAU CAGCAGUGUG AAGACCAUGC CAUUAAGCUA  V  R  T   P  Y  K   Y  H  H  Y   Q  Q  C   E  D  H   A  I  K  L                            nsp2      3760       3770       3780       3790       3800       3810AGCAUGUUGA CCAAGAAAGC AUGUCUGCAU CUGAAUCCCG GCGGAACCUG UGUCAGCAUA  S  M  L   T  K  K   A  C  L  H   L  N  P   G  G  T   C  V  S  I                            nsp2      3820       3830       3840       3850       3860       3870GGUUAUGGUU ACGCUGACAG GGCCAGCGAA AGCAUCAUUG GUGCUAUAGC GCGGCAGUUC  G  Y  G   Y  A  D   R  A  S  E   S  I  I   G  A  I   A  R  Q  F                            nsp2      3880       3890       3900       3910       3920       3930AAGUUUUCCC GAGUAUGCAA ACCGAAAUCC UCACUUGAGG AGACGGAAGU UCUGUUUGUA  K  F  S   R  V  C   K  P  K  S   S  L  E   E  T  E   V  L  F  V                            nsp2      3940       3950       3960       3970       3980       3990UUCAUUGGGU ACGAUCGCAA GGCCCGUACG CACAAUCCUU ACAAGCUAUC AUCAACCUUG  F  I  G   Y  D  R   K  A  R  T   H  N  P   Y  K  L   S  S  T  L                            nsp2      4000       4010       4020       4030 4032ACCAACAUUU AUACAGGUUC CAGACUCCAC GAAGCCGGAU GU  T  N  I   Y  T  G   S  R  L H    E  A  G   C                            nsp2      4042       4052       4062       4072       4082       4092GCACCCUCAU AUCAUGUGGU GCGAGGGGAU AUUGCCACGG CCACCGAAGG AGUGAUUAUA  A  P  S   Y  H  V   V  R  G  D   I  A  T   A  T  E   G  V  I  I                            nsp3      4102       4112       4122       4132       4142       4152AAUGCUGCUA ACAGCAAAGG ACAACCUGGC GGAGGGGUGU GCGGAGCGCU GUAUAAGAAA  N  A  A   N  S  K   G  Q  P  G   G  G  V   C  G  A   L  Y  K  K                            nsp3      4162       4172       4182       4192       4202       4212UUCCCGGAAA GUUUCGAUUU ACAGCCGAUC GAAGUAGGAA AAGCGCGACU GGUCAAAGGU  F  P  E   S  F  D   L  Q  P  I   E  V  G   K  A  R   L  V  K  G                            nsp3      4222       4232       4242       4252       4262       4272GCAGCUAAAC AUAUCAUUCA UGCCGUAGGA CCAAACUUCA ACAAAGUUUC GGAGGUUGAA  A  A  K   H  I  I   H  A  V  G   P  N  F   N  K  V   S  E  V  E                            nsp3      4282       4292       4302       4312       4322       4332GGUGACAAAC AGUUGGCAGA GGCUUAUGAG UCCAUCGCUA AGAUUGUCAA CGAUAACAAU  G  D  K   Q  L  A   E  A  Y  E   S  I  A   K  I  V   N  D  N  N                            nsp3      4342       4352       4362       4372       4382       4392UACAAGUCAG UAGCGAUUCC ACUGUUGUCC ACCGGCAUCU UUUCCGGGAA CAAAGAUCGA  Y  K  S   V  A  I   P  L  L  S   T  G  I   F  S  G   N  K  D  R                            nsp3      4402       4412       4422       4432       4442       4452CUAACCCAAU CAUUGAACCA UUUGCUGACA GCUUUAGACA CCACUGAUGC AGAUGUAGCC  L  T  Q   S  L  N   H  L  L  T   A  L  D   T  T  D   A  D  V  A                            nsp3      4462       4472       4482       4492       4502       4512AUAUACUGCA GGGACAAGAA AUGGGAAAUG ACUCUCAAGG AAGCAGUGGC UAGGAGAGAA  I  Y  C   R  D  K   K  W  E  M   T  L  K   E  A  V   A  R  R  E                            nsp3      4522       4532       4542       4552       4562       4572GCAGUGGAGG AGAUAUGCAU AUCCGACGAU UCUUCAGUGA CAGAACCUGA UGCAGAGCUG  A  V  E   E  I  C   I  S  D  D   S  S  V   T  E  P   D  A  E  L                            nsp3      4582       4592       4602       4612       4622       4632GUGAGGGUGC AUCCCAAGAG UUCUUUGGCU GGAAGGAAGG GCUACAGCAC AAGCGAUGGC  V  R  V   H  P  K   S  S  L  A   G  R  K   G  Y  S   T  S  D  G                            nsp3      4642       4652       4662       4672       4682       4692AAAACUUUCU CAUAUUUGGA AGGGACCAAG UUUCACCAGG CGGCCAAGGA UAUAGCAGAA  K  T  F   S  Y  L   E  G  T  K   F  H  Q   A  A  K   D  I  A  E                            nsp3      4702       4712       4722       4732       4742       4752AUUAAUGCCA UGUGGCCCGU UGCAACGGAG GCCAAUGAGC AGGUAUGCAU GUAUAUCCUC  I  N  A   M  W  P   V  A  T  E   A  N  E   Q  V  C   M  Y  I  L                            nsp3      4762       4772       4782       4792       4802       4812GGAGAAAGCA UGAGCAGUAU UAGGUCGAAA UGCCCCGUCG AGGAGUCGGA AGCCUCCACA  G  E  S   M  S  S   I  R  S  K   C  P  V   E  E  S   E  A  S  T                            nsp3      4822       4832       4842       4852       4862       4872CCACCUAGCA CGCUGCCUUG CUUGUGCAUC CAUGCCAUGA CUCCAGAAAG AGUACAGCGC  P  P  S   T  L  P   C  L  C  I   H  A  M   T  P  E   R  V  Q  R                            nsp3      4882       4892       4912       4922       4932CUAAAAGCCU CACGUCCAGA ACAAAUUACU GUGUGCUCAU CCUUUCCAUU GCCGAAGUAU  L  K  A   S  R  P   E  Q  I  T   V  C  S   S  F  P   L  P  K  Y                            nsp3      4942       4952       4962       4972       4982       4992AGAAUCACUG GUGUGCAGAA GAUCCAAUGC UCCCAGCCUA UAUUGUUCUC ACCGAAAGUG  R  I  T   G  V  Q   K  I  Q  C   S  Q  P   I  L  F   S  P  K  V                            nsp3      5002       5012       5022       5032       5042       5052CCUGCGUAUA UUCAUCCAAG GAAGUAUCUC GUGGAAACAC CACCGGUAGA CGAGACUCCG  P  A  Y   I  H  P   R  K  Y  L   V  E  T   P  P  V   D  E  T  P                            nsp3      5062       5072       5082       5092       5102       5112GAGCCAUCGG CAGAGAACCA AUCCACAGAG GGGACACCUG AACAACCACC ACUUAUAACC  E  P  S   A  E  N   Q  S  T  E   G  T  P   E  Q  P   P  L  I  T                            nsp3      5122       5132       5142       5152       5162       5172GAGGAUGAGA CCAGGACUAG AACGCCUGAG CCGAUCAUCA UCGAAGAAGA AGAAGAAGAU  E  D  E   T  R  T   R  T  P  E   P  I  I   I  E  E   E  E  E  D                            nsp3      5182       5192       5202       5212       5222       5232AGCAUAAGUU UGCUGUCAGA UGGCCCGACC CACCAGGUGC UGCAAGUCGA GGCAGACAUU  S  I  S   L  L  S   D  G  P  T   H  Q  V   L  Q  V   E  A  D  I                            nsp3      5242       5252       5262       5272       5282       5292CACGGGCCGC CCUCUGUAUC UAGCUCAUCC UGGUCCAUUC CUCAUGCAUC CGACUUUGAU  H  G  P   P  S  V   S  S  S  S   W  S  I   P  H  A   S  D  E  D                            nsp3      5302       5312       5322       5332       5342       5352GUGGACAGUU UAUCCAUACU UGACACCCUG GAGGGAGCUA GCGUGACCAG CGGGGCAACG  V  D  S   L  S  I   L  D  T  L   E  G  A   S  V  T   S  G  A  T                            nsp3      5362       5372       5382       5392       5402       5412UCAGCCGAGA CUAACUCUUA CUUCGCAAAG AGUAUGGAGU UUCUGGCGCG ACCGGUGCCU  S  A  E   T  N  S   Y  F  A  K   S  M  E   F  L  A   R  P  V  P                            nsp3      5422       5432       5442       5452       5462       5472GCGCCUCGAA CAGUAUUCAG GAACCCUCCA CAUCCCGCUC CGCGCACAAG AACACCGUCA  A  P  R   T  V  F   R  N  P  P   H  P  A   P  R  T   R  T  P  S                            nsp3      5482       5492       5502       5512       5522       5532CUUGCACCCA GCAGGGCCUG CUCCAGAACC AGCCUAGUUU CCACCCCGCC AGGCGUGAAU  L  A  P   S  R  A   C  S  R  T   S  L  V   S  T  P   P  G  V  N                            nsp3      5542       5552       5562       5572       5582       5592AGGGUGAUCA CUAGAGAGGA GCUCGAAGCG CUUACCCCGU CACGCACUCC UAGCAGGUCG  R  V  I   T  R  E   E  L  E  A   L  T  P   S  R  T   P  S  R  S                            nsp3      5602       5612       5622       5632       5642       5652GUCUCCAGAA CCAGCCUGGU CUCCAACCCG CCAGGCGUAA AUAGGGUGAU UACAAGAGAG  V  S  R   T  S  L   V  S  N  P   P  G  V   N  R  V   I  T  R  E                            nsp3      5662       5672       5682       5692       5702 5703GAGUUUGAGG CGUUCGUAGC ACAACAACAA UGACGGUUUG AUGCGGGUGC A  E  F  E   A  F  V   A  Q  Q  Q   *  R  F   D  A  G   A                            nsp3      5713       5723       5733       5743       5753       5763UACAUCUUUU CCUCCGACAC CGGUCAAGGG CAUUUACAAC AAAAAUCAGU AAGGCAAACG  Y  I  F   S  S  D   T  G  Q  G   H  L  Q   Q  K  S   V  R  Q  T                            nsp4      5773       5783       5793       5803       5813       5823GUGCUAUCCG AAGUGGUGUU GGAGAGGACC GAAUUGGAGA UUUCGUAUGC CCCGCGCCUC  V  L  S   E  V  V   L  E  R  T   E  L  E   I  S  Y   A  P  R  L                            nsp4      5833       5843       5853       5863       5873       5883GACCAAGAAA AAGAAGAAUU ACUACGCAAG AAAUUACAGU UAAAUCCCAC ACCUGCUAAC  D  Q  E   K  E  E   L  L  R  K   K  L  Q   L  N  P   T  P  A  N                            nsp4      5893       5903       5913       5923       5933       5943AGAAGCAGAU ACCAGUCCAG GAAGGUGGAG AACAUGAAAG CCAUAACAGC UAGACGUAUU  R  S  R   Y  Q  S   R  K  V  E   N  M  K   A  I  T   A  R  R  I                            nsp4      5953       5963       5973       5983       5993       6003CUGCAAGGCC UAGGGCAUUA UUUGAAGGCA GAAGGAAAAG UGGAGUGCUA CCGAACCCUG  L  Q  G   L  G  H   Y  L  K  A   E  G  K   V  E  C   Y  R  T  L                            nsp4      6013       6023       6033       6043       6053       6063CAUCCUGUUC CUUUGUAUUC AUCUAGUGUG AACCGUGCCU UUUCAAGCCC CAAGGUCGCA  H  P  V   P  L  Y   S  S  S  V   N  R  A   F  S  S   P  K  V  A                            nsp4      6073       6083       6093       6103       6113       6123GUGGAAGCCU GUAACGCCAU GUUGAAAGAG AACUUUCCGA CUGUGGCUUC UUACUGUAUU  V  E  A   C  N  A   M  L  K  E   N  F  P   T  V  A   S  Y  C  I                            nsp4      6133       6143       6153       6163       6173       6183AUUCCAGAGU ACGAUGCCUA UUUGGACAUG GUUGACGGAG CUUCAUGCUG CUUAGACACU  I  P  E   Y  D  A   Y  L  D  M   V  D  G   A  S  C   C  L  D  T                            nsp4      6193       6203       6213       6223       6233       6243GCCAGUUUUU GCCCUGCAAA GCUGCGCAGC UUUCCAAAGA AACACUCCUA UUUGGAACCC  A  S  F   C  P  A   K  L  R  S   F  P  K   K  H  S   Y  L  E  P                            nsp4      6253       6263       6273       6283       6293       6303ACAAUACGAU CGGCAGUGCC UUCAGCGAUC CAGAACACGC UCCAGAACGU CCUGGCAGCU  T  I  R   S  A  V   P  S  A  I   Q  N  T   L  Q  N   V  L  A  A                            nsp4      6313       6323       6333       6343       6353       6363GCCACAAAAA GAAAUUGCAA UGUCACGCAA AUGAGAGAAU UGCCCGUAUU GGAUUCGGCG  A  T  K   R  N  C   N  V  T  Q   M  R  E   L  P  V   L  D  S  A                            nsp4      6373       6383       6393       6403       6413       6423GCCUUUAAUG UGGAAUGCUU CAAGAAAUAU GCGUGUAAUA AUGAAUAUUG GGAAACGUUU  A  F  N   V  E  C   F  K  K  Y   A  C  N   N  E  Y   W  E  T  F                            nsp4      6433       6443       6453       6463       6473       6483AAAGAAAACC CCAUCAGGCU UACUGAAGAA AACGUGGUAA AUUACAUUAC CAAAUUAAAA  K  E  N   P  I  R   L  T  E  E   N  V  V   N  Y  I   T  K  L  K                            nsp4      6493       6503       6513       6523       6533       6543GGACCAAAAG CUGCUGCUCU UUUUGCGAAG ACACAUAAUU UGAAUAUGUU GCAGGACAUA  G  P  K   A  A  A   L  F  A  K   T  H  N   L  N  M   L  Q  D  I                            nsp4      6553       6563       6573       6583       6593       6603CCAAUGGACA GGUUUGUAAU GGACUUAAAG AGAGACGUGA AAGUGACUCC AGGAACAAAA  P  M  D   R  F  V   M  D  L  K   R  D  V   K  V  T   P  G  T  K                            nsp4      6613       6623       6633       6643       6653       6663CAUACUGAAG AACGGCCCAA GGUACAGGUG AUCCAGGCUG CCGAUCCGCU AGCAACAGCG  H  T  E   E  R  P   K  V  Q  V   I  Q  A   A  D  P   L  A  T  A                            nsp4      6673       6683       6693       6703       6713       6723UAUCUGUGCG GAAUCCACCG AGAGCUGGUU AGGAGAUUAA AUGCGGUCCU GCUUCCGAAC  Y  L  C   G  I  H   R  E  L  V   R  R  L   N  A  V   L  L  P  N                            nsp4      6733       6743       6753       6763       6773       6783AUUCAUACAC UGUUUGAUAU GUCGGCUGAA GACUUUGACG CUAUUAUAGC CGAGCACUUC  I  H  T   L  E  D   M  S  A  E   D  F  D   A  I  I   A  E  H  F                            nsp4      6793       6803       6813       6823       6833       6843CAGCCUGGGG AUUGUGUUCU GGAAACUGAC AUCGCGUCGU UUGAUAAAAG UGAGGACGAC  Q  P  G   D  C  V   L  E  T  D   I  A  S   F  D  K   S  E  D  D                            nsp4      6853       6863       6873       6883       6893       6903GCCAUGGCUC UGACCGCGUU AAUGAUUCUG GAAGACUUAG GUGUGGACGC AGAGCUGUUG  A  M  A   L  T  A   L  M  I  L   E  D  L   G  V  D   A  E  L  L                            nsp4      6913       6923       6933       6943       6953       6963ACGCUGAUUG AGGCGGCUUU CGGCGAAAUU UCAUCAAUAC AUUUGCCCAC UAAAACUAAA  T  L  I   E  A  A   F  G  E  I   S  S  I   H  L  P   T  K  T  K                            nsp4      6973       6983       6993       7003       7013       7023UUUAAAUUCG GAGCCAUGAU GAAAUCUGGA AUGUUCCUCA CACUGUUUGU GAACACAGUC  F  K  F   G  A  M   M  K  S  G   M  F  L   T  L  F   V  N  T  V                            nsp4      7033       7043       7053       7063       7073       7083AUUAACAUUG UAAUCGCAAG CAGAGUGUUG AGAGAACGGC UAACCGGAUC ACCAUGUGCA  I  N  I   V  I  A   S  R  V  L   R  E  R   L  T  G   S  P  C  A                            nsp4      7093       7103       7113       7123       7133       7143GCAUUCAUUG GAGAUGACAA UAUCGUGAAA GGAGUCAAAU CGGACAAAUU AAUGGCAGAC  A  F  I   G  D  D   N  I  V  K   G  V  K   S  D  K   L  M  A  D                            nsp4      7153       7163       7173       7183       7193       7203AGGUGCGCCA CCUGGUUGAA UAUGGAAGUC AAGAUUAUAG AUGCUGUGGU GGGCGAGAAA  R  C  A   T  W  L   N  M  E  V   K  I  I   D  A  V   V  G  E  K                            nsp4      7213       7223       7233       7243       7253       7263GCGCCUUAUU UCUGUGGAGG GUUUAUUUUG UGUGACUCCG UGACCGGCAC AGCGUGCCGU  A  P  Y   F  C  G   G  E  I  L   C  D  S   V  T  G   T  A  C  R                            nsp4      7273       7283       7293       7303       7313       7323GUGGCAGACC CCCUAAAAAG GCUGUUUAAG CUAGGCAAAC CUCUGGCAGC AGACGAUGAA  V  A  D   P  L  K   R  L  F  K   L  G  K   P  L  A   A  D  D  E                            nsp4      7333       7343       7353       7363       7373       7383CAUGAUGAUG ACAGGAGAAG GGCAUUGCAU GAGGAGUCAA CACGCUGGAA CCGAGUGGGU  H  D  D   D  R  R   R  A  L  H   E  E  S   T  R  W   N  R  V  G                            nsp4      7393       7403 7      413       7423       7433       7443AUUCUUUCAG AGCUGUGCAA GGCAGUAGAA UCAAGGUAUG AAACCGUAGG AACUUCCAUC  I  L  S   E  L  C   K  A  V  E   S  R  Y   E  T  V   G  T  S  I                            nsp4      7453       7463       7473       7483       7493       7503AUAGUUAUGG CCAUGACUAC UCUAGCUAGC AGUGUUAAAU CAUUCAGCUA CCUGAGAGGG  I  V  M   A  M  T   T  L  A  S   S  V  K   S  F  S   Y  L  R  G                            nsp4       7513       7523 7527GCCCCUAUAA CUCUCUACGG CUAA   A  P  I   T  L  Y   G  *                            nsp4      7537       7547       7557       7567 7568CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAGACUAG U                          virUTR      7578       7588       7598       7608       7618       7628AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein      7638       7648       7658       7668       7678       7688AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein      7698       7708       7718       7728       7738       7748AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein      7758       7768       7778       7788       7798       7808AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein      7818       7828       7838       7848       7858       7868AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein      7878       7888       7898       7908       7918       7928AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein      7938       7948       7958       7968       7978       7988AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein      7998       8008       8018       8028       8038       8048CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein      8058       8068       8078       8088       8098       8108UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein      8118       8128       8138       8148       8158       8168GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein      8178       8188       8198       8208       8218       8228UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein      8238      8248       8258       8268       8278       8288UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein      8298       8308       8318       8328       8338       8348CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein      8358       8368       8378       8388       8398       8408GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein      8418       8428       8438       8448       8458       8468GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein      8478       8488       8498       8508       8518       8528UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      8538       8548       8558       8568       8578       8588CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      8598       8608       8618       8628       8638       8648GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      8658       8668       8678       8688       8698       8708UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      8718       8728       8738       8748       8758       8768GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      8778       8788       8798       8808       8818       8828GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      8838       8848       8858       8868       8878       8888UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      8898       8908       8918       8928       8938       8948UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      8958       8968       8978       8988       8998       9008CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      9018       9028       9038       9048       9058       9068AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      9078       9088       9098       9108       9118       9128AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      9138       9148       9158       9168       9178       9188CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      9198       9208       9218       9228       9238       9248UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      9258       9268       9278       9288       9298       9308CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      9318       9328       9338       9348       9358       9368ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      9378       9388       9398       9408       9418       9428GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      9438       9448       9458       9468       9478       9488CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      9498       9508       9518       9528       9538       9548AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      9558       9568       9578       9588       9598       9608GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      9618       9628       9638       9648       9658       9668CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      9678       9688       9698       9708       9718       9728GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      9738       9748       9758       9768       9778       9788UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      9798       9808       9818       9828       9838       9848UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      9858       9868       9878       9888       9898       9908ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      9918       9928       9938       9948       9958       9968GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      9978       9988       9998      10008      10018      10028AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein     10038      10048      10058      10068      10078      10088CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein     10098      10108      10118      10128      10138      10148CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein     10158      10168      10178      10188      10198      10208CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein     10218      10228      10238      10248      10258      10268ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein     10278      10288      10298      10308      10318      10328CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein     10338      10348      10358      10368      10378      10388AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein     10398      10408      10418      10428      10438      10448ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein     10458      10468      10478      10488      10498      10508ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein     10518      10528      10538      10548      10558      10568CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein     10578      10588      10598      10608      10618      10628CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein     10638      10648      10658      10668      10678      10688UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein     10698      10708      10718      10728      10738      10748GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein     10758      10768      10778      10788      10798      10808GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein     10818      10828      10838      10848      10858      10868AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein     10878      10888      10898      10908      10918      10928CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein     10938      10948      10958      10968      10978      10988UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein     10998      11008      11018      11028      11038      11048CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein     11058      11068      11078      11088      11098      11108UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein     11118      11128      11138      11148      11158      11168AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein     11178      11188      11198      11208      11218      11228CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein     11238      11248      11258      11268      11278      11288AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein     11298      11308      11318      11328      11338      11348UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein     11358      11368      11378      11388      11393UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein     11403      11413      11423      11433      11443      11453CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element     11463      11473      11483      11493      11503      11513AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element     11523      11533      11543      11553      11563      11573UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element     11583      11593      11603      11613      11623      11633CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element     11643      11653      11663      11673      11683      11693GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCGCGGCC GCAUGAAUAC                          FI element     11703      11713      11723      11733      11743      11753AGCAGCAAUU GGCAAGCUGC UUACAUAGAA CUCGCGGCGA UUGGCAUGCC GCCUUAAAAU                          FI element     11763      11773      11783      11793      11803    11807UUUUAUUUUA UUUUUUCUUU UCUUUUCCGA AUCGGAUUUU GUUUUUAAUA UUUC                          FI element     11817      11827      11837      11847      11857      11867AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)     11877      11887      11897      11907      11917AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40    45GAUGGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA                           5′UTR        55         65         75         85         95        105AUGGAGAAAG UUCACGUUGA CAUCGAGGAA GACAGCCCAU UCCUCAGAGC UUUGCAGCGG  M  E  K   V  H  V   D  I  E  E   D  S  P   F  L  R   A  L  Q  R                            nsp1       115        125        135        145        155        165AGCUUCCCGC AGUUUGAGGU AGAAGCCAAG CAGGUCACUG AUAAUGACCA UGCUAAUGCC  S  F  P   Q  F  E   V  E  A  K   Q  V  T   D  N  D   H  A  N  A                            nsp1       175        185        195        205        215        225AGAGCGUUUU CGCAUCUGGC UUCAAAACUG AUCGAAACGG AGGUGGACCC AUCCGACACG  R  A  F   S  H  L   A  S  K  L  I  E  T    E  V  D   P  S  D  T                            nsp1       235        245        255        265        275        285AUCCUUGACA UUGGAAGUGC GCCCGCCCGC AGAAUGUAUU CUAAGCACAA GUAUCAUUGU  I  L  D   I  G  S   A  P  A  R  R  M  Y    S  K  H   K  Y  H  C                            nsp1       295        305        315        325        335        345AUCUGUCCGA UGAGAUGUGC GGAAGAUCCG GACAGAUUGU AUAAGUAUGC AACUAAGCUG I  C  P    M  R  C   A  E  D  P   D  R  L   Y  K  Y   A  T  K  L                            nsp1       355        365        375        385        395        405AAGAAAAACU GUAAGGAAAU AACUGAUAAG GAAUUGGACA AGAAAAUGAA GGAGCUCGCC  K  K  N   C  K  E   I  T  D  K   E  L  D   K  K  M   K  E  L  A                            nsp1       415        425        435        445        455        465GCCGUCAUGA GCGACCCUGA CCUGGAAACU GAGACUAUGU GCCUCCACGA CGACGAGUCG  A  V  M   S  D  P   D  L  E  T   E  T  M   C  L  H   D  D  E  S                            nsp1       475        485        495        505        515        525UGUCGCUACG AAGGGCAAGU CGCUGUUUAC CAGGAUGUAU ACGCGGUUGA CGGACCGACA  C  R  Y   E  G  Q   V  A  V  Y   Q  D  V   Y  A  V   D  G  P  T                            nsp1       535        545        555        565        575        585AGUCUCUAUC ACCAAGCCAA UAAGGGAGUU AGAGUCGCCU ACUGGAUAGG CUUUGACACC  S  L  Y   H  Q  A   N  K  G  V   R  V  A   Y  W  I   G  F  D  T                            nsp1       595        605        615        625        635        645ACCCCUUUUA UGUUUAAGAA CUUGGCUGGA GCAUAUCCAU CAUACUCUAC CAACUGGGCC  T  P  F   M  F  K   N  L  A  G   A  Y  P   S  Y  S   T  N  W  A                            nsp1       655        665        675        685        695        705GACGAAACCG UGUUAACGGC UCGUAACAUA GGCCUAUGCA GCUCUGACGU UAUGGAGCGG  D  E  T   V  L  T   A  R  N  I   G  L  C   S  S  D   V  M  E  R                            nsp1       715        725        735        745        755        765UCACGUAGAG GGAUGUCCAU UCUUAGAAAG AAGUAUUUGA AACCAUCCAA CAAUGUUCUA  S  R  R   G  M  S   I  L  R  K   K  Y  L   K  P  S   N  N  V  L                            nsp1       775        785        795        805        815        825UUCUCUGUUG GCUCGACCAU CUACCACGAA AAGAGGGACU UACUGAGGAG CUGGCACCUG  F  S  V   G  S  T   I  Y  H  E   K  R  D   L  L  R   S  W  H  L                            nsp1       835        845        855        865        875        885CCGUCUGUAU UUCACUUACG UGGCAAGCAA AAUUACACAU GUCGGUGUGA GACUAUAGUU  P  S  V   F  H  L   R  G  K  Q   N  Y  T   C  R  C   E  T  I  V                            nsp1       895        905        915        925        935        945AGUUGCGACG GGUACGUCGU UAAAAGAAUA GCUAUCAGUC CAGGCCUGUA UGGGAAGCCU  S  C  D   G  Y  V   V  K  R  I   A  I  S   P  G  L   Y  G  K  P                            nsp1       955        965        975        985        995       1005UCAGGCUAUG CUGCUACGAU GCACCGCGAG GGAUUCUUGU GCUGCAAAGU GACAGACACA  S  G  Y   A  A  T   M  H  R  E   G  E  L   C  C  K   V  T  D  T                            nsp1      1015       1025       1035       1045       1055       1065UUGAACGGGG AGAGGGUCUC UUUUCCCGUG UGCACGUAUG UGCCAGCUAC AUUGUGUGAC  L  N  G   E  R  V   S  F  P  V   C  T  Y   V  P  A   T  L  C  D                            nsp1      1075       1085       1095       1105       1115       1125CAAAUGACUG GCAUACUGGC AACAGAUGUC AGUGCGGACG ACGCGCAAAA ACUGCUGGUU  Q  M  T   G  I  L   A  T  D  V   S  A  D   D  A  Q   K  L  L  V                            nsp1      1135       1145       1155       1165       1175       1185GGGCUCAACC AGCGUAUAGU CGUCAACGGU CGCACCCAGA GAAACACCAA UACCAUGAAA  G  L  N   Q  R  I   V  V  N  G   R  T  Q   R  N  T   N  T  M  K                            nsp1      1198       1205       1215       1225       1235       1245AAUUACCUUU UGCCCGUAGU GGCCCAGGCA UUUGCUAGGU GGGCAAAGGA AUAUAAGGAA  N  Y  L   L  P  V   V  A  Q  A   F  A  R   W  A  K   E  Y  K  E                            nsp1      1255       1265       1275       1285       1295       1305GAUCAAGAAG AUGAAAGGCC ACUAGGACUA CGAGAUAGAC AGUUAGUCAU GGGGUGUUGU  D  Q  E   D  E  R   P  L  G  L   R  D  R   Q  L  V   M  G  C  C                            nsp1      1315       1325       1335       1345       1355       1365UGGGCUUUUA GAAGGCACAA GAUAACAUCU AUUUAUAAGC GCCCGGAUAC CCAAACCAUC  W  A  F   R  R  H   K  I  T  S   I  Y  K   R  P  D  T  Q  T  I                            nsp1      1375       1385       1395       1405       1415       1425AUCAAAGUGA ACAGCGAUUU CCACUCAUUC GUGCUGCCCA GGAUAGGCAG UAACACAUUG  I  K  V   N  S  D   F  H  S  F   V  L  P   R  I  G   S  N  T  L                            nsp1      1435       1445       1455       1465       1475       1485GAGAUCGGGC UGAGAACAAG AAUCAGGAAA AUGUUAGAGG AGCACAAGGA GCCGUCACCU  E  I  G   L  R  T   R  I  R  K   M  L  E   E  H  K   E  P  S  P                            nsp1      1495       1505       1515       1525       1535       1545CUCAUUACCG CCGAGGACGU ACAAGAAGCU AAGUGCGCAG CCGAUGAGGC UAAGGAGGUG  L  I  T   A  E  D   V  Q  E  A   K  C  A   A  D  E   A  K  E  V                            nsp1      1555       1565       1575       1585       1595       1605CGUGAAGCCG AGGAGUUGCG CGCAGCUCUA CCACCUUUGG CAGCUGAUGU UGAGGAGCCC  R  E  A   E  E  L   R  A  A  L   P  P  L   A  A  D   V  E  E  P                            nsp1      1615       1625       1635       1645       1650ACUCUGGAAG CCGAUGUCGA CUUGAUGUUA CAAGAGGCUG GGGCC  T  L  E   A  D  V   D  L  M  L   Q  E  A   G  A                            nsp1      1660       1670       1680       1690       1700       1710GGCUCAGUGG AGACACCUCG UGGCUUGAUA AAGGUUACCA GCUACGCUGG CGAGGACAAG  G  S  V   E  T  P   R  G  L  I   K  V  T   S  Y  A   G  E  D  K                            nsp2      1720       1730       1740       1750       1760       1770AUCGGCUCUU ACGCUGUGCU UUCUCCGCAG GCUGUACUCA AGAGUGAAAA AUUAUCUUGC  I  G  S   Y  A  V   L  S  P  Q   A  V  L   K  S  E   K  L  S  C                            nsp2      1780       1790       1800       1810       1820       1830AUCCACCCUC UCGCUGAACA AGUCAUAGUG AUAACACACU CUGGCCGAAA AGGGCGUUAU  I  H  P   L  A  E   Q  V  I  V   I  T  H   S  G  R   K  G  R  Y                            nsp2      1840       1850       1860       1870       1880       1890GCCGUGGAAC CAUACCAUGG UAAAGUAGUG GUGCCAGAGG GACAUGCAAU ACCCGUCCAG  A  V  E   P  Y  H   G  K  V  V   V  P  E   G  H  A   I  P  V  Q                            nsp2      1900       1910       1920       1930       1940       1950GACUUUCAAG CUCUGAGUGA AAGUGCCACC AUUGUGUACA ACGAACGUGA GUUCGUAAAC  D  F  Q   A  L  S   E  S  A  T   I  V  Y   N  E  R   E  F  V  N                            nsp2      1960       1970       1980       1990       2000       2010AGGUACCUGC ACCAUAUUGC CACACAUGGA GGAGCGCUGA ACACUGAUGA AGAAUAUUAC  R  Y  L   H  H  I   A  T  H  G   G  A  L   N  T  D   E  E  Y  Y                            nsp2      2020       2030       2040       2050       2060       2070AAAACUGUCA AGCCCAGCGA GCACGACGGC GAAUACCUGU ACGACAUCGA CAGGAAACAG  K  T  V   K  P  S   E  H  D  G   E  Y  L   Y  D  I   D  R  K  Q                            nsp2      2080       2090       2100       2110       2120       2130UGCGUCAAGA AAGAGCUAGU CACUGGGCUA GGGCUCACAG GCGAGCUGGU CGAUCCUCCC  C  V  K   K  E  L   V  T  G  L   G  L  T   G  E  L   V  D  P  P                            nsp2      2140       2150       2160       2170       2180       2190UUCCAUGAAU UCGCCUACGA GAGUCUGAGA ACACGACCAG CCGCUCCUUA CCAAGUACCA  F  H  E   F  A  Y   E  S  L  R   T  R  P   A  A  P   Y  Q  V  P                            nsp2      2200       2210       2220       2230       2240       2250ACCAUAGGGG UGUAUGGCGU GCCAGGAUCA GGCAAGUCUG GCAUCAUUAA AAGCGCAGUC  T  I  G   V  Y  G   V  P  G  S   G  K  S   G  I  I   K  S  A  V                            nsp2      2260       2270       2280       2290       2300       2310ACCAAAAAAG AUCUAGUGGU GAGCGCCAAG AAAGAAAACU GUGCAGAAAU UAUAAGGGAC  T  K  K   D  L  V   V  S  A  K   K  E  N   C  A  E   I  I  R  D                            nsp2      2320       2330       2340       2350       2360       2370GUCAAGAAAA UGAAAGGGCU GGACGUCAAU GCCAGAACUG UGGACUCAGU GCUCUUGAAU  V  K  K   M  K  G   L  D  V  N   A  R  T   V  D  S   V  L  L  N                            nsp2      2380       2390       2400       2410       2420       2430GGAUGCAAAC ACCCCGUAGA GACCCUGUAU AUUGACGAGG CUUUUGCUUG UCAUGCAGGU  G  C  K   H  P  V   E  T  L  Y   I  D  E   A  F  A   C  H  A  G                            nsp2      2440       2450       2460       2470       2480       2490ACUCUCAGAG CGCUCAUAGC CAUUAUAAGA CCUAAAAAGG CAGUGCUCUG CGGAGAUCCC  T  L  R   A  L  I   A  I  I  R   P  K  K   A  V  L   C  G  D  P                            nsp2      2500       2510       2520       2530       2540       2550AAACAGUGCG GUUUUUUUAA CAUGAUGUGC CUGAAAGUGC AUUUUAACCA CGAGAUUUGC  K  Q  C   G  E  E   N  M  M  C   L  K  V   H  F  N   H  E  I  C                            nsp2      2560       2570       2580       2590       2600       2610ACACAAGUCU UCCACAAAAG CAUCUCUCGC CGUUGCACUA AAUCUGUGAC UUCGGUCGUC  T  Q  V   F  H  K   S  I  S  R   R  C  T   K  S  V   T  S  V  V                            nsp2      2620       2630       2640       2650       2660       2670UCAACCUUGU UUUACGACAA AAAAAUGAGA ACGACGAAUC CGAAAGAGAC UAAGAUUGUG  S  T  L   F  Y  D   K  K  M  R   T  T  N   P  K  E   T  K  I  V                            nsp2      2680       2690       2700       2710       2720       2730AUUGACACUA CCGGCAGUAC CAAACCUAAG CAGGACGAUC UCAUUCUCAC UUGUUUCAGA  I  D  T   T  G  S   T  K  P  K   Q  D  D   L  I  L   T  C  F  R                            nsp2      2740       2750       2760       2770       2780       2790GGGUGGGUGA AGCAGUUGCA AAUAGAUUAC AAAGGCAACG AAAUAAUGAC GGCAGCUGCC  G  W  V   K  Q  L   Q  I  D  Y   K  G  N   E  I  M   T  A  A  A                            nsp2      2800       2810       2820       2830       2840       2850UCUCAAGGGC UGACCCGUAA AGGUGUGUAU GCCGUUCGGU ACAAGGUGAA UGAAAAUCCU  S  Q  G   L  T  R   K  G  V  Y   A  V  R   Y  K  V   N  E  N  P                            nsp2      2860       2870       2880       2890       2900       2910CUGUACGCAC CCACCUCAGA ACAUGUGAAC GUCCUACUGA CCCGCACGGA GGACCGCAUC  L  Y  A   P  T  S   E  H  V  N   V  L  L   T  R  T   E  D  R  I                            nsp2      2920       2930       2940       2950       2960       2970GUGUGGAAAA CACUAGCCGG CGACCCAUGG AUAAAAACAC UGACUGCCAA GUACCCUGGG  V  W  K   T  L  A   G  D  P  W   I  K  T   L  T  A   K  Y  P  G                            nsp2      2980       2990       3000       3010       3020       3030AAUUUCACUG CCACGAUAGA GGAGUGGCAA GCAGAGCAUG AUGCCAUCAU GAGGCACAUC  N  F  T   A  T  I   E  E  W  Q   A  E  H   D  A  I   M  R  H  I                            nsp2      3040       3050       3060       3070       3080       3090UUGGAGAGAC CGGACCCUAC CGACGUCUUC CAGAAUAAGG CAAACGUGUG UUGGGCCAAG  L  E  R   P  D  P   T  D  V  F   Q  N  K   A  N  V   C  W  A  K                            nsp2      3100       3110       3120       3130       3140       3150GCUUUAGUGC CGGUGCUGAA GACCGCUGGC AUAGACAUGA CCACUGAACA AUGGAACACU  A  L  V   P  V  L   K  T  A  G   I  D  M   T  T  E   Q  W  N  T                            nsp2      3160       3170       3180       3190       3200       3210GUGGAUUAUU UUGAAACGGA CAAAGCUCAC UCAGCAGAGA UAGUAUUGAA CCAACUAUGC  V  D  Y   F  E  T   D  K  A  H   S  A  E   I  V  L   N  Q  L  C                            nsp2      3220       3230       3240       3250       3260       3270GUGAGGUUCU UUGGACUCGA UCUGGACUCC GGUCUAUUUU CUGCACCCAC UGUUCCGUUA  V  R  F   F  G  L   D  L  D  S   G  L  F   S  A  P   T  V  P  L                            nsp2      3280       3290       3300       3310       3320       3330UCCAUUAGGA AUAAUCACUG GGAUAACUCC CCGUCGCCUA ACAUGUACGG GCUGAAUAAA  S  I  R   N  N  H   W  D  N  S   P  S  P   N  M  Y   G  L  N  K                            nsp2      3340       3350       3360       3370       3380       3390GAAGUGGUCC GUCAGCUCUC UCGCAGGUAC CCACAACUGC CUCGGGCAGU UGCCACUGGU  E  V  V   R  Q  L   S  R  R  Y   P  Q  L   P  R  A   V  A  T  G                            nsp2      3400       3410       3420       3430       3440       3450AGAGUCUAUG ACAUGAACAC UGGUACACUG CGCAAUUAUG AUCCGCGCAU AAACCUAGUA  R  V  Y   D  M  N   T  G  T  L   R  N  Y   D  P  R   I  N  L  V                            nsp2      3460       3470       3480       3490       3500       3510CCUGUAAACA GAAGACUGCC UCAUGCUUUA GUCCUCCACC AUAAUGAACA CCCACAGAGU  P  V  N   R  R  L   P  H  A  L   V  L  H   H  N  E   H  P  Q  S                            nsp2      3520       3530       3540       3550       3560       3570GACUUUUCUU CAUUCGUCAG CAAAUUGAAG GGCAGAACUG UCCUGGUGGU CGGGGAAAAG  D  F  S   S  F  V   S  K  L  K   G  R  T   V  L  V   V  G  E  K                            nsp2      3580       3590       3600       3610       3620       3630UUGUCCGUCC CAGGCAAAAU GGUUGACUGG UUGUCAGACC GGCCUGAGGC UACCUUCAGA  L  S  V   P  G  K   M  V  D  W   L  S  D   R  P  E   A  T  E  R                            nsp2      3640       3650       3660       3670       3680       3690GCUCGGCUGG AUUUAGGCAU CCCAGGUGAU GUGCCCAAAU AUGACAUAAU AUUUGUUAAU  A  R  L   D  L  G   I  P  G  D   V  P  K   Y  D  I   I  F  V  N                            nsp2      3700       3710       3720       3730       3740       3750GUGAGGACCC CAUAUAAAUA CCAUCACUAU CAGCAGUGUG AAGACCAUGC CAUUAAGCUA  V  R  T   P  Y  K   Y  H  H  Y   Q  Q  C   E  D  H   A  I  K  L                            nsp2      3760       3770       3780       3790       3800       3810AGCAUGUUGA CCAAGAAAGC AUGUCUGCAU CUGAAUCCCG GCGGAACCUG UGUCAGCAUA  S  M  L   T  K  K   A  C  L  H   L  N  P   G  G  T   C  V  S  I                            nsp2      3820       3830       3840       3850       3860       3870GGUUAUGGUU ACGCUGACAG GGCCAGCGAA AGCAUCAUUG GUGCUAUAGC GCGGCAGUUC  G  Y  G   Y  A  D   R  A  S  E   S  I  I   G  A  I   A  R  Q  F                            nsp2      3880       3890       3900       3910       3920       3930AAGUUUUCCC GAGUAUGCAA ACCGAAAUCC UCACUUGAGG AGACGGAAGU UCUGUUUGUA  K  F  S   R  V  C   K  P  K  S   S  L  E   E  T  E   V  L  F  V                            nsp2      3940       3950       3960       3970       3980       3990UUCAUUGGGU ACGAUCGCAA GGCCCGUACG CACAAUCCUU ACAAGCUAUC AUCAACCUUG  F  I  G   Y  D  R   K  A  R  T   H  N  P   Y  K  L   S  S  T  L                            nsp2      4000       4010       4020       4030 4032ACCAACAUUU AUACAGGUUC CAGACUCCAC GAAGCCGGAU GU  T  N  I   Y  T  G   S  R  L H    E  A  G   C                            nsp2      4042       4052       4062       4072       4082       4092GCACCCUCAU AUCAUGUGGU GCGAGGGGAU AUUGCCACGG CCACCGAAGG AGUGAUUAUA  A  P  S   Y  H  V   V  R  G  D   I  A  T   A  T  E   G  V  I  I                            nsp3      4102       4112       4122       4132       4142       4152AAUGCUGCUA ACAGCAAAGG ACAACCUGGC GGAGGGGUGU GCGGAGCGCU GUAUAAGAAA  N  A  A   N  S  K   G  Q  P  G   G  G  V   C  G  A   L  Y  K  K                            nsp3      4162       4172       4182       4192       4202       4212UUCCCGGAAA GUUUCGAUUU ACAGCCGAUC GAAGUAGGAA AAGCGCGACU GGUCAAAGGU  F  P  E   S  F  D   L  Q  P  I   E  V  G   K  A  R   L  V  K  G                            nsp3      4222       4232       4242       4252       4262       4272GCAGCUAAAC AUAUCAUUCA UGCCGUAGGA CCAAACUUCA ACAAAGUUUC GGAGGUUGAA  A  A  K   H  I  I   H  A  V  G   P  N  F   N  K  V   S  E  V  E                            nsp3      4282       4292       4302       4312       4322       4332GGUGACAAAC AGUUGGCAGA GGCUUAUGAG UCCAUCGCUA AGAUUGUCAA CGAUAACAAU  G  D  K   Q  L  A   E  A  Y  E   S  I  A   K  I  V   N  D  N  N                            nsp3      4342       4352       4362       4372       4382       4392UACAAGUCAG UAGCGAUUCC ACUGUUGUCC ACCGGCAUCU UUUCCGGGAA CAAAGAUCGA  Y  K  S   V  A  I   P  L  L  S   T  G  I   F  S  G   N  K  D  R                            nsp3      4402       4412       4422       4432       4442       4452CUAACCCAAU CAUUGAACCA UUUGCUGACA GCUUUAGACA CCACUGAUGC AGAUGUAGCC  L  T  Q   S  L  N   H  L  L  T   A  L  D   T  T  D   A  D  V  A                            nsp3      4462       4472       4482       4492       4502       4512AUAUACUGCA GGGACAAGAA AUGGGAAAUG ACUCUCAAGG AAGCAGUGGC UAGGAGAGAA  I  Y  C   R  D  K   K  W  E  M   T  L  K   E  A  V   A  R  R  E                            nsp3      4522       4532       4542       4552       4562       4572GCAGUGGAGG AGAUAUGCAU AUCCGACGAU UCUUCAGUGA CAGAACCUGA UGCAGAGCUG  A  V  E   E  I  C   I  S  D  D   S  S  V   T  E  P   D  A  E  L                            nsp3      4582       4592       4602       4612       4622       4632GUGAGGGUGC AUCCCAAGAG UUCUUUGGCU GGAAGGAAGG GCUACAGCAC AAGCGAUGGC  V  R  V   H  P  K   S  S  L  A   G  R  K   G  Y  S   T  S  D  G                            nsp3      4642       4652       4662       4672       4682       4692AAAACUUUCU CAUAUUUGGA AGGGACCAAG UUUCACCAGG CGGCCAAGGA UAUAGCAGAA  K  T  F   S  Y  L   E  G  T  K   F  H  Q   A  A  K   D  I  A  E                            nsp3      4702       4712       4722       4732       4742       4752AUUAAUGCCA UGUGGCCCGU UGCAACGGAG GCCAAUGAGC AGGUAUGCAU GUAUAUCCUC  I  N  A   M  W  P   V  A  T  E   A  N  E   Q  V  C   M  Y  I  L                            nsp3      4762       4772       4782       4792       4802       4812GGAGAAAGCA UGAGCAGUAU UAGGUCGAAA UGCCCCGUCG AGGAGUCGGA AGCCUCCACA  G  E  S   M  S  S   I  R  S  K   C  P  V   E  E  S   E  A  S  T                            nsp3      4822       4832       4842       4852       4862       4872CCACCUAGCA CGCUGCCUUG CUUGUGCAUC CAUGCCAUGA CUCCAGAAAG AGUACAGCGC  P  P  S   T  L  P   C  L  C  I   H  A  M   T  P  E   R  V  Q  R                            nsp3      4882       4892       4902       4912       4922       4932CUAAAAGCCU CACGUCCAGA ACAAAUUACU GUGUGCUCAU CCUUUCCAUU GCCGAAGUAU  L  K  A   S  R  P   E  Q  I  T   V  C  S   S  F  P   L  P  K  Y                            nsp3      4942       4952       4962       4972       4982       4992AGAAUCACUG GUGUGCAGAA GAUCCAAUGC UCCCAGCCUA UAUUGUUCUC ACCGAAAGUG  R  I  T   G  V  Q   K  I  Q  C   S  Q  P   I  L  F   S  P  K  V                            nsp3      5002       5012       5022       5032       5042       5052CCUGCGUAUA UUCAUCCAAG GAAGUAUCUC GUGGAAACAC CACCGGUAGA CGAGACUCCG  P  A  Y   I  H  P   R  K  Y  L   V  E  T   P  P  V   D  E  T  P                            nsp3      5062       5072       5082       5092       5102       5112GAGCCAUCGG CAGAGAACCA AUCCACAGAG GGGACACCUG AACAACCACC ACUUAUAACC  E  P  S   A  E  N   Q  S  T  E   G  T  P   E  Q  P   P  L  I  T                            nsp3      5122       5132       5142       5152       5162       5172GAGGAUGAGA CCAGGACUAG AACGCCUGAG CCGAUCAUCA UCGAAGAAGA AGAAGAAGAU  E  D  E   T  R  T   R  T  P  E   P  I  I   I  E  E   E  E  E  D                            nsp3      5182       5192       5202       5212       5222       5232AGCAUAAGUU UGCUGUCAGA UGGCCCGACC CACCAGGUGC UGCAAGUCGA GGCAGACAUU  S  I  S   L  L  S   D  G  P  T   H  Q  V   L  Q  V   E  A  D  I                            nsp3      5242       5252       5262       5272       5282       5292CACGGGCCGC CCUCUGUAUC UAGCUCAUCC UGGUCCAUUC CUCAUGCAUC CGACUUUGAU  H  G  P   P  S  V   S  S  S  S   W  S  I   P  H  A   S  D  E  D                            nsp3      5302       5312       5322       5332       5342       5352GUGGACAGUU UAUCCAUACU UGACACCCUG GAGGGAGCUA GCGUGACCAG CGGGGCAACG  V  D  S   L  S  I   L  D  T  L   E  G  A   S  V  T   S  G  A  T                            nsp3      5362       5372       5382       5392       5402       5412UCAGCCGAGA CUAACUCUUA CUUCGCAAAG AGUAUGGAGU UUCUGGCGCG ACCGGUGCCU  S  A  E   T  N  S   Y  F  A  K   S  M  E   F  L  A   R  P  V  P                            nsp3      5422       5432       5442       5452       5462       5472GCGCCUCGAA CAGUAUUCAG GAACCCUCCA CAUCCCGCUC CGCGCACAAG AACACCGUCA  A  P  R   T  V  F   R  N  P  P   H  P  A   P  R  T   R  T  P  S                            nsp3      5482       5492       5502       5512       5522       5532CUUGCACCCA GCAGGGCCUG CUCCAGAACC AGCCUAGUUU CCACCCCGCC AGGCGUGAAU  L  A  P   S  R  A   C  S  R  T   S  L  V   S  T  P   P  G  V  N                            nsp3      5542       5552       5562       5572       5582       5592AGGGUGAUCA CUAGAGAGGA GCUCGAAGCG CUUACCCCGU CACGCACUCC UAGCAGGUCG  R  V  I   T  R  E   E  L  E  A   L  T  P   S  R  T   P  S  R  S                            nsp3      5602       5612       5622       5632       5642       5652GUCUCCAGAA CCAGCCUGGU CUCCAACCCG CCAGGCGUAA AUAGGGUGAU UACAAGAGAG  V  S  R   T  S  L   V  S  N  P   P  G  V   N  R  V   I  T  R  E                            nsp3      5662       5672       5682       5692       5702 5703GAGUUUGAGG CGUUCGUAGC ACAACAACAA UGACGGUUUG AUGCGGGUGC A  E  F  E   A  F  V   A  Q  Q  Q   *  R  F   D  A  G   A                            nsp3      5713       5723       5733       5743       5753       5763UACAUCUUUU CCUCCGACAC CGGUCAAGGG CAUUUACAAC AAAAAUCAGU AAGGCAAACG  Y  I  F   S  S  D   T  G  Q  G   H  L  Q   Q  K  S   V  R  Q  T                            nsp4      5773       5783       5793       5803       5813       5823GUGCUAUCCG AAGUGGUGUU GGAGAGGACC GAAUUGGAGA UUUCGUAUGC CCCGCGCCUC  V  L  S   E  V  V   L  E  R  T   E  L  E   I  S  Y   A  P  R  L                            nsp4      5833       5843       5853       5863       5873       5883GACCAAGAAA AAGAAGAAUU ACUACGCAAG AAAUUACAGU UAAAUCCCAC ACCUGCUAAC  D  Q  E   K  E  E   L  L  R  K   K  L  Q   L  N  P   T  P  A  N                            nsp4      5893       5903       5913       5923       5933       5943AGAAGCAGAU ACCAGUCCAG GAAGGUGGAG AACAUGAAAG CCAUAACAGC UAGACGUAUU  R  S  R   Y  Q  S   R  K  V  E   N  M  K   A  I  T   A  R  R  I                            nsp4      5953       5963       5973       5983       5993       6003CUGCAAGGCC UAGGGCAUUA UUUGAAGGCA GAAGGAAAAG UGGAGUGCUA CCGAACCCUG  L  Q  G   L  G  H   Y  L  K  A   E  G  K   V  E  C   Y  R  T  L                            nsp4      6013       6023       6033       6043       6053       6063CAUCCUGUUC CUUUGUAUUC AUCUAGUGUG AACCGUGCCU UUUCAAGCCC CAAGGUCGCA  H  P  V   P  L  Y   S  S  S  V   N  R  A   F  S  S   P  K  V  A                            nsp4      6073       6083       6093       6103       6113       6123GUGGAAGCCU GUAACGCCAU GUUGAAAGAG AACUUUCCGA CUGUGGCUUC UUACUGUAUU  V  E  A   C  N  A   M  L  K  E   N  F  P   T  V  A   S  Y  C  I                            nsp4      6133       6143       6153       6163       6173       6183AUUCCAGAGU ACGAUGCCUA UUUGGACAUG GUUGACGGAG CUUCAUGCUG CUUAGACACU  I  P  E   Y  D  A   Y  L  D  M   V  D  G   A  S  C   C  L  D  T                            nsp4      6193       6203       6213       6223       6233       6243GCCAGUUUUU GCCCUGCAAA GCUGCGCAGC UUUCCAAAGA AACACUCCUA UUUGGAACCC  A  S  F   C  P  A   K  L  R  S   F  P  K   K  H  S   Y  L  E  P                            nsp4      6253       6263       6273       6283       6293       6303ACAAUACGAU CGGCAGUGCC UUCAGCGAUC CAGAACACGC UCCAGAACGU CCUGGCAGCU  T  I  R   S  A  V   P  S  A  I   Q  N  T   L  Q  N   V  L  A  A                            nsp4      6313       6323       6333       6343       6353       6363GCCACAAAAA GAAAUUGCAA UGUCACGCAA AUGAGAGAAU UGCCCGUAUU GGAUUCGGCG  A  T  K   R  N  C   N  V  T  Q   M  R  E   L  P  V   L  D  S  A                            nsp4      6373       6383       6393       6403       6413       6423GCCUUUAAUG UGGAAUGCUU CAAGAAAUAU GCGUGUAAUA AUGAAUAUUG GGAAACGUUU  A  F  N   V  E  C   F  K  K  Y   A  C  N   N  E  Y   W  E  T  F                            nsp4      6433       6443       6453       6463       6473       6483AAAGAAAACC CCAUCAGGCU UACUGAAGAA AACGUGGUAA AUUACAUUAC CAAAUUAAAA  K  E  N   P  I  R   L  T  E  E   N  V  V   N  Y  I   T  K  L  K                            nsp4      6493       6503       6513       6523       6533       6543GGACCAAAAG CUGCUGCUCU UUUUGCGAAG ACACAUAAUU UGAAUAUGUU GCAGGACAUA  G  P  K   A  A  A   L  F  A  K   T  H  N   L  N  M   L  Q  D  I                            nsp4      6553       6563       6573       6583       6593       6603CCAAUGGACA GGUUUGUAAU GGACUUAAAG AGAGACGUGA AAGUGACUCC AGGAACAAAA  P  M  D   R  F  V   M  D  L  K   R  D  V   K  V  T   P  G  T  K                            nsp4      6613       6623       6633       6643       6653       6663CAUACUGAAG AACGGCCCAA GGUACAGGUG AUCCAGGCUG CCGAUCCGCU AGCAACAGCG  H  T  E   E  R  P   K  V  Q  V   I  Q  A   A  D  P   L  A  T  A                            nsp4      6673       6683       6693       6703       6713       6723UAUCUGUGCG GAAUCCACCG AGAGCUGGUU AGGAGAUUAA AUGCGGUCCU GCUUCCGAAC  Y  L  C   G  I  H   R  E  L  V   R  R  L   N  A  V   L  L  P  N                            nsp4      6733       6743       6753       6763       6773       6783AUUCAUACAC UGUUUGAUAU GUCGGCUGAA GACUUUGACG CUAUUAUAGC CGAGCACUUC  I  H  T   L  E  D   M  S  A  E   D  F  D   A  I  I   A  E  H  F                            nsp4      6793       6803       6813       6823       6833       6843CAGCCUGGGG AUUGUGUUCU GGAAACUGAC AUCGCGUCGU UUGAUAAAAG UGAGGACGAC  Q  P  G   D  C  V   L  E  T  D   I  A  S   F  D  K   S  E  D  D                            nsp4      6853       6863       6873       6883       6893       6903GCCAUGGCUC UGACCGCGUU AAUGAUUCUG GAAGACUUAG GUGUGGACGC AGAGCUGUUG  A  M  A   L  T  A   L  M  I  L   E  D  L   G  V  D   A  E  L  L                            nsp4      6913       6923       6933       6943       6953       6963ACGCUGAUUG AGGCGGCUUU CGGCGAAAUU UCAUCAAUAC AUUUGCCCAC UAAAACUAAA  T  L  I   E  A  A   F  G  E  I   S  S  I   H  L  P   T  K  T  K                            nsp4      6973       6983       6993       7003       7013       7023UUUAAAUUCG GAGCCAUGAU GAAAUCUGGA AUGUUCCUCA CACUGUUUGU GAACACAGUC  F  K  F   G  A  M   M  K  S  G   M  F  L   T  L  F   V  N  T  V                            nsp4      7033       7043       7053       7063       7073       7083AUUAACAUUG UAAUCGCAAG CAGAGUGUUG AGAGAACGGC UAACCGGAUC ACCAUGUGCA  I  N  I   V  I  A   S  R  V  L   R  E  R   L  T  G   S  P  C  A                            nsp4      7093       7103       7113       7123       7133       7143GCAUUCAUUG GAGAUGACAA UAUCGUGAAA GGAGUCAAAU CGGACAAAUU AAUGGCAGAC  A  F  I   G  D  D   N  I  V  K   G  V  K   S  D  K   L  M  A  D                            nsp4      7153       7163       7173       7183       7193       7203AGGUGCGCCA CCUGGUUGAA UAUGGAAGUC AAGAUUAUAG AUGCUGUGGU GGGCGAGAAA  R  C  A   T  W  L   N  M  E  V   K  I  I   D  A  V   V  G  E  K                            nsp4      7213       7223       7233       7243       7253       7263GCGCCUUAUU UCUGUGGAGG GUUUAUUUUG UGUGACUCCG UGACCGGCAC AGCGUGCCGU  A  P  Y   F  C  G   G  E  I  L   C  D  S   V  T  G   T  A  C  R                            nsp4      7273       7283       7293       7303       7313       7323GUGGCAGACC CCCUAAAAAG GCUGUUUAAG CUAGGCAAAC CUCUGGCAGC AGACGAUGAA  V  A  D   P  L  K   R  L  F  K   L  G  K   P  L  A   A  D  D  E                            nsp4      7333       7343       7353       7363       7373       7383CAUGAUGAUG ACAGGAGAAG GGCAUUGCAU GAGGAGUCAA CACGCUGGAA CCGAGUGGGU  H  D  D   D  R  R   R  A  L  H   E  E  S   T  R  W   N  R  V  G                            nsp4      7393       7403 7      413       7423       7433       7443AUUCUUUCAG AGCUGUGCAA GGCAGUAGAA UCAAGGUAUG AAACCGUAGG AACUUCCAUC  I  L  S   E  L  C   K  A  V  E   S  R  Y   E  T  V   G  T  S  I                            nsp4      7453       7463       7473       7483       7493       7503AUAGUUAUGG CCAUGACUAC UCUAGCUAGC AGUGUUAAAU CAUUCAGCUA CCUGAGAGGG  I  V  M   A  M  T   T  L  A  S   S  V  K   S  F  S   Y  L  R  G                            nsp4       7513       7523 7527GCCCCUAUAA CUCUCUACGG CUAA   A  P  I   T  L  Y   G  *                            nsp4      7537       7547       7557       7567 7568CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAGACUAG U                          virUTR      7578       7588       7598       7608       7618       7628AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGAA UUUGACAACA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein      7638       7648       7658       7668       7678       7688AGAACACAGC UGCCACCAGC UUAUACAAAU UCUUUUACCA GAGGAGUGUA UUAUCCUGAU  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein      7698       7708       7718       7728       7738       7748AAAGUGUUUA GAUCUUCUGU GCUGCACAGC ACACAGGACC UGUUUCUGCC AUUUUUUAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein      7758       7768       7778       7788       7798       7808AAUGUGACAU GGUUUCAUGC AAUUCAUGUG UCUGGAACAA AUGGAACAAA AAGAUUUGAU  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein      7818       7828       7838       7848       7858       7868AAUCCUGUGC UGCCUUUUAA UGAUGGAGUG UAUUUUGCUU CAACAGAAAA GUCAAAUAUU  N  P  V   L  P  F   N  D  G  V   Y  E  A   S  T  E   K  S  N  I                          S protein      7878       7888       7898       7908       7918       7928AUUAGAGGAU GGAUUUUUGG AACAACACUG GAUUCUAAAA CACAGUCUCU GCUGAUUGUG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein      7938       7948       7958       7968       7978       7988AAUAAUGCAA CAAAUGUGGU GAUUAAAGUG UGUGAAUUUC AGUUUUGUAA UGAUCCUUUU  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein      7998       8008       8018       8028       8038       8048CUGGGAGUGU AUUAUCACAA AAAUAAUAAA UCUUGGAUGG AAUCUGAAUU UAGAGUGUAU  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S protein      8058       8068       8078       8088       8098       8108UCCUCUGCAA AUAAUUGUAC AUUUGAAUAU GUGUCUCAGC CUUUUCUGAU GGAUCUGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   P  F  L   M  D  L  E                          S protein      8118       8128       8138       8148       8158       8168GGAAAACAGG GCAAUUUUAA AAAUCUGAGA GAAUUUGUGU UUAAAAAUAU UGAUGGAUAU  G  K  Q   G  N  F   K  N  L  R   E  F  V   F  K  N   I  D  G  Y                          S protein      8178       8188       8198       8208       8218       8228UUUAAAAUUU AUUCUAAACA CACACCAAUU AAUUUAGUGA GAGAUCUGCC UCAGGGAUUU  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  D  L   P  Q  G  F                          S protein      8238      8248       8258       8268       8278       8288UCUGCUCUGG AACCUCUGGU GGAUCUGCCA AUUGGCAUUA AUAUUACAAG AUUUCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S protein      8298       8308       8318       8328       8338       8348CUGCUGGCUC UGCACAGAUC UUAUCUGACA CCUGGAGAUU CUUCUUCUGG AUGGACAGCC  L  L  A   L  H  R   S  Y  L  T   P  G  D   S  S  S   G  W  T  A                          S protein      8358       8368       8378       8388       8398       8408GGAGCUGCAG CUUAUUAUGU GGGCUAUCUG CAGCCAAGAA CAUUUCUGCU GAAAUAUAAU  G  A  A   A  Y  Y   V  G  Y  L   Q  P  R   T  F  L   L  K  Y  N                          S protein      8418       8428       8438       8448       8458       8468GAAAAUGGAA CAAUUACAGA UGCUGUGGAU UGUGCUCUGG AUCCUCUGUC UGAAACAAAA  E  N  G   T  I  T   D  A  V  D   C  A  L   D  P  L   S  E  T  K                          S protein      8478       8488       8498       8508       8518       8528UGUACAUUAA AAUCUUUUAC AGUGGAAAAA GGCAUUUAUC AGACAUCUAA UUUUAGAGUG  C  T  L   K  S  F   T  V  E  K   G  I  Y   Q  T  S   N  F  R  V                          S protein      8538       8548       8558       8568       8578       8588CAGCCAACAG AAUCUAUUGU GAGAUUUCCA AAUAUUACAA AUCUGUGUCC AUUUGGAGAA  Q  P  T   E  S  I   V  R  F  P   N  I  T   N  L  C   P  F  G  E                          S protein      8598       8608       8618       8628       8638       8648GUGUUUAAUG CAACAAGAUU UGCAUCUGUG UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU  V  F  N   A  T  R   F  A  S  V   Y  A  W   N  R  K   R  I  S  N                          S protein      8658       8668       8678       8688       8698       8708UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU AGUGCUUCUU UUUCCACAUU UAAAUGUUAU  C  V  A   D  Y  S   V  L  Y  N   S  A  S   F  S  T   F  K  C  Y                          S protein      8718       8728       8738       8748       8758       8768GGAGUGUCUC CAACAAAAUU AAAUGAUUUA UGUUUUACAA AUGUGUAUGC UGAUUCUUUU  G  V  S   P  T  K   L  N  D  L   C  F  T   N  V  Y   A  D  S  F                          S protein      8778       8788       8798       8808       8818       8828GUGAUCAGAG GUGAUGAAGU GAGACAGAUU GCCCCCGGAC AGACAGGAAA AAUUGCUGAU  V  I  R   G  D  E   V  R  Q  I   A  P  G   Q  T  G   K  I  A  D                          S protein      8838       8848       8858       8868       8878       8888UACAAUUACA AACUGCCUGA UGAUUUUACA GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU  Y  N  Y   K  L  P   D  D  F  T   G  C  V   I  A  W   N  S  N  N                          S protein      8898       8908       8918       8928       8938       8948UUAGAUUCUA AAGUGGGAGG AAAUUACAAU UAUCUGUACA GACUGUUUAG AAAAUCAAAU  L  D  S   K  V  G   G  N  Y  N   Y  L  Y   R  L  F   R  K  S  N                          S protein      8958       8968       8978       8988       8998       9008CUGAAACCUU UUGAAAGAGA UAUUUCAACA GAAAUUUAUC AGGCUGGAUC AACACCUUGU  L  K  P   P  E  R   D  I  S  T   E  I  Y   Q  A  G   S  T  P  C                          S protein      9018       9028       9038       9048       9058       9068AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU CCAUUACAGA GCUAUGGAUU UCAGCCAACC  N  G  V   E  G  F   N  C  Y  F   P  L  Q   S  Y  G   F  Q  P  T                          S protein      9078       9088       9098       9108       9118       9128AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG GUGGUGCUGU CUUUUGAACU GCUGCAUGCA  N  G  V   G  Y  Q   P  Y  R  V   V  V  L   S  F  E   L  L  H  A                          S protein      9138       9148       9158       9168       9178       9188CCUGCAACAG UGUGUGGACC UAAAAAAUCU ACAAAUUUAG UGAAAAAUAA AUGUGUGAAU  P  A  T   V  C  G   P  K  K  S   T  N  L   V  K  N   K  C  V  N                          S protein      9198       9208       9218       9228       9238       9248UUUAAUUUUA AUGGAUUAAC AGGAACAGGA GUGCUGACAG AAUCUAAUAA AAAAUUUCUG  F  N  F   N  G  L   T  G  T  G   V  L  T   E  S  N   K  K  F  L                          S protein      9258       9268       9278       9288       9298       9308CCUUUUCAGC AGUUUGGCAG AGAUAUUGCA GAUACCACAG AUGCAGUGAG AGAUCCUCAG  P  F  Q   Q  F  G   R  D  I  A   D  T  T   D  A  V   R  D  P  Q                          S protein      9318       9328       9338       9348       9358       9368ACAUUAGAAA UUCUGGAUAU UACACCUUGU UCUUUUGGGG GUGUGUCUGU GAUUACACCU  T  L  E   I  L  D   I  T  P  C   S  F  G   G  V  S   V  I  T  P                          S protein      9378       9388       9398       9408       9418       9428GGAACAAAUA CAUCUAAUCA GGUGGCUGUG CUGUAUCAGG AUGUGAAUUG UACAGAAGUG  G  T  N   T  S  N   Q  V  A  V   L  Y  Q   D  V  N   C  T  E  V                          S protein      9438       9448       9458       9468       9478       9488CCAGUGGCAA UUCAUGCAGA UCAGCUGACA CCAACAUGGA GAGUGUAUUC UACAGGAUCU  P  V  A   I  H  A   D  Q  L  T   P  T  W   R  V  Y   S  T  G  S                          S protein      9498       9508       9518       9528       9538       9548AAUGUGUUUC AGACAAGAGC AGGAUGUCUG AUUGGAGCAG AACAUGUGAA UAAUUCUUAU  N  V  F   Q  T  R   A  G  C  L   I  G  A   E  H  V   N  N  S  Y                          S protein      9558       9568       9578       9588       9598       9608GAAUGUGAUA UUCCAAUUGG AGCAGGCAUU UGUGCAUCUU AUCAGACACA GACAAAUUCC  E  C  D   I  P  I   G  A  G  I   C  A  S   Y  Q  T   Q  T  N  S                          S protein      9618       9628       9638       9648       9658       9668CCAAGGAGAG CAAGAUCUGU GGCAUCUCAG UCUAUUAUUG CAUACACCAU GUCUCUGGGA  P  R  R   A  R  S   V  A  S  Q   S  I  I   A  Y  T   M  S  L  G                          S protein      9678       9688       9698       9708       9718       9728GCAGAAAAUU CUGUGGCAUA UUCUAAUAAU UCUAUUGCUA UUCCAACAAA UUUUACCAUU  A  E  N   S  V  A   Y  S  N  N   S  I  A   I  P  T   N  F  T  I                          S protein      9738       9748       9758       9768       9778       9788UCUGUGACAA CAGAAAUUUU ACCUGUGUCU AUGACAAAAA CAUCUGUGGA UUGUACCAUG  S  V  T   T  E  I   L  P  V  S   M  T  K   T  S  V   D  C  T  M                          S protein      9798       9808       9818       9828       9838       9848UACAUUUGUG GAGAUUCUAC AGAAUGUUCU AAUCUGCUGC UGCAGUAUGG AUCUUUUUGU  Y  I  C   G  D  S   T  E  C  S   N  L  L   L  Q  Y   G  S  F  C                          S protein      9858       9868       9878       9888       9898       9908ACACAGCUGA AUAGAGCUUU AACAGGAAUU GCUGUGGAAC AGGAUAAAAA UACACAGGAA  T  Q  L   N  R  A   L  T  G  I   A  V  E   Q  D  K   N  T  Q  E                          S protein      9918       9928       9938       9948       9958       9968GUGUUUGCUC AGGUGAAACA GAUUUACAAA ACACCACCAA UUAAAGAUUU UGGAGGAUUU  V  F  A   Q  V  K   Q  I  Y  K  T  P  P    I  K  D   F  G  G  F                          S protein      9978       9988       9998      10008      10018      10028AAUUUUAGCC AGAUUCUGCC UGAUCCUUCU AAACCUUCUA AAAGAUCUUU UAUUGAAGAU  N  F  S   Q  I  L   P  D  P  S   K  P  S   K  R  S   F  I  E  D                          S protein     10038      10048      10058      10068      10078      10088CUGCUGUUUA AUAAAGUGAC ACUGGCAGAU GCAGGAUUUA UUAAACAGUA UGGAGAUUGC  L  L  F   N  K  V   T  L  A  D   A  G  F   I  K  Q   Y  G  D  C                          S protein     10098      10108      10118      10128      10138      10148CUGGGUGAUA UUGCUGCAAG AGAUCUGAUU UGUGCUCAGA AAUUUAAUGG ACUGACAGUG  L  G  D   I  A  A   R  D  L  I   C  A  Q   K  F  N   G  L  T  V                          S protein     10158      10168      10178      10188      10198      10208CUGCCUCCUC UGCUGACAGA UGAAAUGAUU GCUCAGUACA CAUCUGCUUU ACUGGCUGGA  L  P  P   L  L  T   D  E  M  I   A  Q  Y   T  S  A   L  L  A  G                          S protein     10218      10228      10238      10248      10258      10268ACAAUUACAA GCGGAUGGAC AUUUGGAGCU GGAGCUGCUC UGCAGAUUCC UUUUGCAAUG  T  I  T   S  G  W   T  F  G  A   G  A  A   L  Q  I   P  F  A  M                          S protein     10278      10288      10298      10308      10318      10328CAGAUGGCUU ACAGAUUUAA UGGAAUUGGA GUGACACAGA AUGUGUUAUA UGAAAAUCAG  Q  M  A   Y  R  F   N  G  I  G   V  T  Q   N  V  L   Y  E  N  Q                          S protein     10338      10348      10358      10368      10378      10388AAACUGAUUG CAAAUCAGUU UAAUUCUGCA AUUGGCAAAA UUCAGGAUUC UCUGUCUUCU  K  L  I   A  N  Q   F  N  S  A   I  G   K  I  Q  D   S  L  S  S                          S protein     10398      10408      10418      10428      10438      10448ACAGCUUCUG CUCUGGGAAA ACUGCAGGAU GUGGUGAAUC AGAAUGCACA GGCACUGAAU  T  A  S   A  L  G   K  L  Q  D   V  V  N   Q  N  A   Q  A  L  N                          S protein     10458      10468      10478      10488      10498      10508ACUCUGGUGA AACAGCUGUC UAGCAAUUUU GGGGCAAUUU CUUCUGUGCU GAAUGAUAUU  T  L  V   K  Q  L   S  S  N  F   G  A  I   S  S  V   L  N  D  I                          S protein     10518      10528      10538      10548      10558      10568CUGUCUAGAC UGGAUCCTCC TGAAGCUGAA GUGCAGAUUG AUAGACUGAU CACAGGAAGA  L  S  R   L  D  P   P  E  A  E   V  Q  I   D  R  L   I  T  G  R                          S protein     10578      10588      10598      10608      10618      10628CUGCAGUCUC UGCAGACUUA UGUGACACAG CAGCUGAUUA GAGCUGCUGA AAUUAGAGCU  L  Q  S   L  Q  T   Y  V  T  Q   Q  L  I   R  A  A   E  I  R  A                          S protein     10638      10648      10658      10668      10678      10688UCUGCUAAUC UGGCUGCUAC AAAAAUGUCU GAAUGUGUGC UGGGACAGUC AAAAAGAGUG  S  A  N   L  A  A   T  K  M  S   E  C  V   L  G  Q   S  K  R  V                          S protein     10698      10708      10718      10728      10738      10748GAUUUUUGUG GAAAAGGAUA UCAUCUGAUG UCUUUUCCAC AGUCUGCUCC ACAUGGAGUG  D  F  C   G  K  G   Y  H  L  M  S  E  P    Q  S  A   P  H  G  V                          S protein     10758      10768      10778      10788      10798      10808GUGUUUUUAC AUGUGACAUA UGUGCCAGCA CAGGAAAAGA AUUUUACCAC AGCACCAGCA  V  F  L   H  V  T   Y  V  P  A   Q  E  K   N  F  T   T  A  P  A                          S protein     10818      10828      10838      10848      10858      10868AUUUGUCAUG AUGGAAAAGC ACAUUUUCCA AGAGAAGGAG UGUUUGUGUC UAAUGGAACA  I  C  H   D  G  K   A  H  F  P   R  E  G   V  F  V   S  N  G  T                          S protein     10878      10888      10898      10908      10918      10928CAUUGGUUUG UGACACAGAG AAAUUUUUAU GAACCUCAGA UUAUUACAAC AGAUAAUACA  H  W  F   V  T  Q   R  N  F  Y   E  P  Q   I  I  T   T  D  N  T                          S protein     10938      10948      10958      10968      10978      10988UUUGUGUCAG GAAAUUGUGA UGUGGUGAUU GGAAUUGUGA AUAAUACAGU GUAUGAUCCA  F  V  S   G  N  C   D  V  V  I   G  I  V   N  N  T   V  Y  D  P                          S protein     10998      11008      11018      11028      11038      11048CUGCAGCCAG AACUGGAUUC UUUUAAAGAA GAACUGGAUA AAUAUUUUAA AAAUCACACA  L  Q  P   E  L  D   S  F  K  E   E  L  D   K  Y  F   K  N  H  T                          S protein     11058      11068      11078      11088      11098      11108UCUCCUGAUG UGGAUUUAGG AGAUAUUUCU GGAAUCAAUG CAUCUGUGGU GAAUAUUCAG  S  P  D   V  D  L   G  D  I  S   G  I  N   A  S  V   V  N  I  Q                          S protein     11118      11128      11138      11148      11158      11168AAAGAAAUUG AUAGACUGAA UGAAGUGGCC AAAAAUCUGA AUGAAUCUCU GAUUGAUCUG  K  E  I   D  R  L   N  E  V  A   K  N  L   N  E  S   L  I  D  L                          S protein     11178      11188      11198      11208      11218      11228CAGGAACUUG GAAAAUAUGA ACAGUACAUU AAAUGGCCUU GGUACAUUUG GCUUGGAUUU  Q  E  L   G  K  Y   E  Q  Y  I   K  W  P   W  Y  I   W  L  G  F                          S protein     11238      11248      11258      11268      11278      11288AUUGCAGGAU UAAUUGCAAU UGUGAUGGUG ACAAUUAUGU UAUGUUGUAU GACAUCAUGU  I  A  G   L  I  A   I  V  M  V   T  I  M   L  C  C   M  T  S  C                          S protein     11298      11308      11318      11328      11338      11348UGUUCUUGUU UAAAAGGAUG UUGUUCUUGU GGAAGCUGUU GUAAAUUUGA UGAAGAUGAU  C  S  C   L  K  G   C  C  S  C   G  S  C   C  K  F   D  E  D  D                          S protein     11358      11368      11378      11388      11393UCUGAACCUG UGUUAAAAGG AGUGAAAUUG CAUUACACAU GAUGA  S  E  P   V  L  K   G  V  K  L   H  Y  T   * *                          S protein     11403      11413      11423      11433      11443      11453CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element     11463      11473      11483      11493      11503      11513AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element     11523      11533      11543      11553      11563      11573UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element     11583      11593      11603      11613      11623      11633CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element     11643      11653      11663      11673      11683      11693GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCGCGGCC GCAUGAAUAC                          FI element     11703      11713      11723      11733      11743      11753AGCAGCAAUU GGCAAGCUGC UUACAUAGAA CUCGCGGCGA UUGGCAUGCC GCCUUAAAAU                          FI element     11763      11773      11783      11793      11803    11807UUUUAUUUUA UUUUUUCUUU UCUUUUCCGA AUCGGAUUUU GUUUUUAAUA UUUC                          FI element     11817      11827      11837      11847      11857      11867AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)     11877      11887      11897      11907      11917AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40    45GAUGGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA                           5′UTR        55         65         75         85         95        105AUGGAGAAAG UUCACGUUGA CAUCGAGGAA GACAGCCCAU UCCUCAGAGC UUUGCAGCGG  M  E  K   V  H  V   D  I  E  E   D  S  P   F  L  R   A  L  Q  R                            nsp1       115        125        135        145        155        165AGCUUCCCGC AGUUUGAGGU AGAAGCCAAG CAGGUCACUG AUAAUGACCA UGCUAAUGCC  S  F  P   Q  F  E   V  E  A  K   Q  V  T   D  N  D   H  A  N  A                            nsp1       175        185        195        205        215        225AGAGCGUUUU CGCAUCUGGC UUCAAAACUG AUCGAAACGG AGGUGGACCC AUCCGACACG  R  A  F   S  H  L   A  S  K  L  I  E  T    E  V  D   P  S  D  T                            nsp1       235        245        255        265        275        285AUCCUUGACA UUGGAAGUGC GCCCGCCCGC AGAAUGUAUU CUAAGCACAA GUAUCAUUGU  I  L  D   I  G  S   A  P  A  R  R  M  Y    S  K  H   K  Y  H  C                            nsp1       295        305        315        325        335        345AUCUGUCCGA UGAGAUGUGC GGAAGAUCCG GACAGAUUGU AUAAGUAUGC AACUAAGCUG I  C  P    M  R  C   A  E  D  P   D  R  L   Y  K  Y   A  T  K  L                            nsp1       355        365        375        385        395        405AAGAAAAACU GUAAGGAAAU AACUGAUAAG GAAUUGGACA AGAAAAUGAA GGAGCUCGCC  K  K  N   C  K  E   I  T  D  K   E  L  D   K  K  M   K  E  L  A                            nsp1       415        425        435        445        455        465GCCGUCAUGA GCGACCCUGA CCUGGAAACU GAGACUAUGU GCCUCCACGA CGACGAGUCG  A  V  M   S  D  P   D  L  E  T   E  T  M   C  L  H   D  D  E  S                            nsp1       475        485        495        505        515        525UGUCGCUACG AAGGGCAAGU CGCUGUUUAC CAGGAUGUAU ACGCGGUUGA CGGACCGACA  C  R  Y   E  G  Q   V  A  V  Y   Q  D  V   Y  A  V   D  G  P  T                            nsp1       535        545        555        565        575        585AGUCUCUAUC ACCAAGCCAA UAAGGGAGUU AGAGUCGCCU ACUGGAUAGG CUUUGACACC  S  L  Y   H  Q  A   N  K  G  V   R  V  A   Y  W  I   G  F  D  T                            nsp1       595        605        615        625        635        645ACCCCUUUUA UGUUUAAGAA CUUGGCUGGA GCAUAUCCAU CAUACUCUAC CAACUGGGCC  T  P  F   M  F  K   N  L  A  G   A  Y  P   S  Y  S   T  N  W  A                            nsp1       655        665        675        685        695        705GACGAAACCG UGUUAACGGC UCGUAACAUA GGCCUAUGCA GCUCUGACGU UAUGGAGCGG  D  E  T   V  L  T   A  R  N  I   G  L  C   S  S  D   V  M  E  R                            nsp1       715        725        735        745        755        765UCACGUAGAG GGAUGUCCAU UCUUAGAAAG AAGUAUUUGA AACCAUCCAA CAAUGUUCUA  S  R  R   G  M  S   I  L  R  K   K  Y  L   K  P  S   N  N  V  L                            nsp1       775        785        795        805        815        825UUCUCUGUUG GCUCGACCAU CUACCACGAA AAGAGGGACU UACUGAGGAG CUGGCACCUG  F  S  V   G  S  T   I  Y  H  E   K  R  D   L  L  R   S  W  H  L                            nsp1       835        845        855        865        875        885CCGUCUGUAU UUCACUUACG UGGCAAGCAA AAUUACACAU GUCGGUGUGA GACUAUAGUU  P  S  V   F  H  L   R  G  K  Q   N  Y  T   C  R  C   E  T  I  V                            nsp1       895        905        915        925        935        945AGUUGCGACG GGUACGUCGU UAAAAGAAUA GCUAUCAGUC CAGGCCUGUA UGGGAAGCCU  S  C  D   G  Y  V   V  K  R  I   A  I  S   P  G  L   Y  G  K  P                            nsp1       955        965        975        985        995       1005UCAGGCUAUG CUGCUACGAU GCACCGCGAG GGAUUCUUGU GCUGCAAAGU GACAGACACA  S  G  Y   A  A  T   M  H  R  E   G  E  L   C  C  K   V  T  D  T                            nsp1      1015       1025       1035       1045       1055       1065UUGAACGGGG AGAGGGUCUC UUUUCCCGUG UGCACGUAUG UGCCAGCUAC AUUGUGUGAC  L  N  G   E  R  V   S  F  P  V   C  T  Y   V  P  A   T  L  C  D                            nsp1      1075       1085       1095       1105       1115       1125CAAAUGACUG GCAUACUGGC AACAGAUGUC AGUGCGGACG ACGCGCAAAA ACUGCUGGUU  Q  M  T   G  I  L   A  T  D  V   S  A  D   D  A  Q   K  L  L  V                            nsp1      1135       1145       1155       1165       1175       1185GGGCUCAACC AGCGUAUAGU CGUCAACGGU CGCACCCAGA GAAACACCAA UACCAUGAAA  G  L  N   Q  R  I   V  V  N  G   R  T  Q   R  N  T   N  T  M  K                            nsp1      1198       1205       1215       1225       1235       1245AAUUACCUUU UGCCCGUAGU GGCCCAGGCA UUUGCUAGGU GGGCAAAGGA AUAUAAGGAA  N  Y  L   L  P  V   V  A  Q  A   F  A  R   W  A  K   E  Y  K  E                            nsp1      1255       1265       1275       1285       1295       1305GAUCAAGAAG AUGAAAGGCC ACUAGGACUA CGAGAUAGAC AGUUAGUCAU GGGGUGUUGU  D  Q  E   D  E  R   P  L  G  L   R  D  R   Q  L  V   M  G  C  C                            nsp1      1315       1325       1335       1345       1355       1365UGGGCUUUUA GAAGGCACAA GAUAACAUCU AUUUAUAAGC GCCCGGAUAC CCAAACCAUC  W  A  F   R  R  H   K  I  T  S   I  Y  K   R  P  D  T  Q  T  I                            nsp1      1375       1385       1395       1405       1415       1425AUCAAAGUGA ACAGCGAUUU CCACUCAUUC GUGCUGCCCA GGAUAGGCAG UAACACAUUG  I  K  V   N  S  D   F  H  S  F   V  L  P   R  I  G   S  N  T  L                            nsp1      1435       1445       1455       1465       1475       1485GAGAUCGGGC UGAGAACAAG AAUCAGGAAA AUGUUAGAGG AGCACAAGGA GCCGUCACCU  E  I  G   L  R  T   R  I  R  K   M  L  E   E  H  K   E  P  S  P                            nsp1      1495       1505       1515       1525       1535       1545CUCAUUACCG CCGAGGACGU ACAAGAAGCU AAGUGCGCAG CCGAUGAGGC UAAGGAGGUG  L  I  T   A  E  D   V  Q  E  A   K  C  A   A  D  E   A  K  E  V                            nsp1      1555       1565       1575       1585       1595       1605CGUGAAGCCG AGGAGUUGCG CGCAGCUCUA CCACCUUUGG CAGCUGAUGU UGAGGAGCCC  R  E  A   E  E  L   R  A  A  L   P  P  L   A  A  D   V  E  E  P                            nsp1      1615       1625       1635       1645       1650ACUCUGGAAG CCGAUGUCGA CUUGAUGUUA CAAGAGGCUG GGGCC  T  L  E   A  D  V   D  L  M  L   Q  E  A   G  A                            nsp1      1660       1670       1680       1690       1700       1710GGCUCAGUGG AGACACCUCG UGGCUUGAUA AAGGUUACCA GCUACGCUGG CGAGGACAAG  G  S  V   E  T  P   R  G  L  I   K  V  T   S  Y  A   G  E  D  K                            nsp2      1720       1730       1740       1750       1760       1770AUCGGCUCUU ACGCUGUGCU UUCUCCGCAG GCUGUACUCA AGAGUGAAAA AUUAUCUUGC  I  G  S   Y  A  V   L  S  P  Q   A  V  L   K  S  E   K  L  S  C                            nsp2      1780       1790       1800       1810       1820       1830AUCCACCCUC UCGCUGAACA AGUCAUAGUG AUAACACACU CUGGCCGAAA AGGGCGUUAU  I  H  P   L  A  E   Q  V  I  V   I  T  H   S  G  R   K  G  R  Y                            nsp2      1840       1850       1860       1870       1880       1890GCCGUGGAAC CAUACCAUGG UAAAGUAGUG GUGCCAGAGG GACAUGCAAU ACCCGUCCAG  A  V  E   P  Y  H   G  K  V  V   V  P  E   G  H  A   I  P  V  Q                            nsp2      1900       1910       1920       1930       1940       1950GACUUUCAAG CUCUGAGUGA AAGUGCCACC AUUGUGUACA ACGAACGUGA GUUCGUAAAC  D  F  Q   A  L  S   E  S  A  T   I  V  Y   N  E  R   E  F  V  N                            nsp2      1960       1970       1980       1990       2000       2010AGGUACCUGC ACCAUAUUGC CACACAUGGA GGAGCGCUGA ACACUGAUGA AGAAUAUUAC  R  Y  L   H  H  I   A  T  H  G   G  A  L   N  T  D   E  E  Y  Y                            nsp2      2020       2030       2040       2050       2060       2070AAAACUGUCA AGCCCAGCGA GCACGACGGC GAAUACCUGU ACGACAUCGA CAGGAAACAG  K  T  V   K  P  S   E  H  D  G   E  Y  L   Y  D  I   D  R  K  Q                            nsp2      2080       2090       2100       2110       2120       2130UGCGUCAAGA AAGAGCUAGU CACUGGGCUA GGGCUCACAG GCGAGCUGGU CGAUCCUCCC  C  V  K   K  E  L   V  T  G  L   G  L  T   G  E  L   V  D  P  P                            nsp2      2140       2150       2160       2170       2180       2190UUCCAUGAAU UCGCCUACGA GAGUCUGAGA ACACGACCAG CCGCUCCUUA CCAAGUACCA  F  H  E   F  A  Y   E  S  L  R   T  R  P   A  A  P   Y  Q  V  P                            nsp2      2200       2210       2220       2230       2240       2250ACCAUAGGGG UGUAUGGCGU GCCAGGAUCA GGCAAGUCUG GCAUCAUUAA AAGCGCAGUC  T  I  G   V  Y  G   V  P  G  S   G  K  S   G  I  I   K  S  A  V                            nsp2      2260       2270       2280       2290       2300       2310ACCAAAAAAG AUCUAGUGGU GAGCGCCAAG AAAGAAAACU GUGCAGAAAU UAUAAGGGAC  T  K  K   D  L  V   V  S  A  K   K  E  N   C  A  E   I  I  R  D                            nsp2      2320       2330       2340       2350       2360       2370GUCAAGAAAA UGAAAGGGCU GGACGUCAAU GCCAGAACUG UGGACUCAGU GCUCUUGAAU  V  K  K   M  K  G   L  D  V  N   A  R  T   V  D  S   V  L  L  N                            nsp2      2380       2390       2400       2410       2420       2430GGAUGCAAAC ACCCCGUAGA GACCCUGUAU AUUGACGAGG CUUUUGCUUG UCAUGCAGGU  G  C  K   H  P  V   E  T  L  Y   I  D  E   A  F  A   C  H  A  G                            nsp2      2440       2450       2460       2470       2480       2490ACUCUCAGAG CGCUCAUAGC CAUUAUAAGA CCUAAAAAGG CAGUGCUCUG CGGAGAUCCC  T  L  R   A  L  I   A  I  I  R   P  K  K   A  V  L   C  G  D  P                            nsp2      2500       2510       2520       2530       2540       2550AAACAGUGCG GUUUUUUUAA CAUGAUGUGC CUGAAAGUGC AUUUUAACCA CGAGAUUUGC  K  Q  C   G  E  E   N  M  M  C   L  K  V   H  F  N   H  E  I  C                            nsp2      2560       2570       2580       2590       2600       2610ACACAAGUCU UCCACAAAAG CAUCUCUCGC CGUUGCACUA AAUCUGUGAC UUCGGUCGUC  T  Q  V   F  H  K   S  I  S  R   R  C  T   K  S  V   T  S  V  V                            nsp2      2620       2630       2640       2650       2660       2670UCAACCUUGU UUUACGACAA AAAAAUGAGA ACGACGAAUC CGAAAGAGAC UAAGAUUGUG  S  T  L   F  Y  D   K  K  M  R   T  T  N   P  K  E   T  K  I  V                            nsp2      2680       2690       2700       2710       2720       2730AUUGACACUA CCGGCAGUAC CAAACCUAAG CAGGACGAUC UCAUUCUCAC UUGUUUCAGA  I  D  T   T  G  S   T  K  P  K   Q  D  D   L  I  L   T  C  F  R                            nsp2      2740       2750       2760       2770       2780       2790GGGUGGGUGA AGCAGUUGCA AAUAGAUUAC AAAGGCAACG AAAUAAUGAC GGCAGCUGCC  G  W  V   K  Q  L   Q  I  D  Y   K  G  N   E  I  M   T  A  A  A                            nsp2      2800       2810       2820       2830       2840       2850UCUCAAGGGC UGACCCGUAA AGGUGUGUAU GCCGUUCGGU ACAAGGUGAA UGAAAAUCCU  S  Q  G   L  T  R   K  G  V  Y   A  V  R   Y  K  V   N  E  N  P                            nsp2      2860       2870       2880       2890       2900       2910CUGUACGCAC CCACCUCAGA ACAUGUGAAC GUCCUACUGA CCCGCACGGA GGACCGCAUC  L  Y  A   P  T  S   E  H  V  N   V  L  L   T  R  T   E  D  R  I                            nsp2      2920       2930       2940       2950       2960       2970GUGUGGAAAA CACUAGCCGG CGACCCAUGG AUAAAAACAC UGACUGCCAA GUACCCUGGG  V  W  K   T  L  A   G  D  P  W   I  K  T   L  T  A   K  Y  P  G                            nsp2      2980       2990       3000       3010       3020       3030AAUUUCACUG CCACGAUAGA GGAGUGGCAA GCAGAGCAUG AUGCCAUCAU GAGGCACAUC  N  F  T   A  T  I   E  E  W  Q   A  E  H   D  A  I   M  R  H  I                            nsp2      3040       3050       3060       3070       3080       3090UUGGAGAGAC CGGACCCUAC CGACGUCUUC CAGAAUAAGG CAAACGUGUG UUGGGCCAAG  L  E  R   P  D  P   T  D  V  F   Q  N  K   A  N  V   C  W  A  K                            nsp2      3100       3110       3120       3130       3140       3150GCUUUAGUGC CGGUGCUGAA GACCGCUGGC AUAGACAUGA CCACUGAACA AUGGAACACU  A  L  V   P  V  L   K  T  A  G   I  D  M   T  T  E   Q  W  N  T                            nsp2      3160       3170       3180       3190       3200       3210GUGGAUUAUU UUGAAACGGA CAAAGCUCAC UCAGCAGAGA UAGUAUUGAA CCAACUAUGC  V  D  Y   F  E  T   D  K  A  H   S  A  E   I  V  L   N  Q  L  C                            nsp2      3220       3230       3240       3250       3260       3270GUGAGGUUCU UUGGACUCGA UCUGGACUCC GGUCUAUUUU CUGCACCCAC UGUUCCGUUA  V  R  F   F  G  L   D  L  D  S   G  L  F   S  A  P   T  V  P  L                            nsp2      3280       3290       3300       3310       3320       3330UCCAUUAGGA AUAAUCACUG GGAUAACUCC CCGUCGCCUA ACAUGUACGG GCUGAAUAAA  S  I  R   N  N  H   W  D  N  S   P  S  P   N  M  Y   G  L  N  K                            nsp2      3340       3350       3360       3370       3380       3390GAAGUGGUCC GUCAGCUCUC UCGCAGGUAC CCACAACUGC CUCGGGCAGU UGCCACUGGU  E  V  V   R  Q  L   S  R  R  Y   P  Q  L   P  R  A   V  A  T  G                            nsp2      3400       3410       3420       3430       3440       3450AGAGUCUAUG ACAUGAACAC UGGUACACUG CGCAAUUAUG AUCCGCGCAU AAACCUAGUA  R  V  Y   D  M  N   T  G  T  L   R  N  Y   D  P  R   I  N  L  V                            nsp2      3460       3470       3480       3490       3500       3510CCUGUAAACA GAAGACUGCC UCAUGCUUUA GUCCUCCACC AUAAUGAACA CCCACAGAGU  P  V  N   R  R  L   P  H  A  L   V  L  H   H  N  E   H  P  Q  S                            nsp2      3520       3530       3540       3550       3560       3570GACUUUUCUU CAUUCGUCAG CAAAUUGAAG GGCAGAACUG UCCUGGUGGU CGGGGAAAAG  D  F  S   S  F  V   S  K  L  K   G  R  T   V  L  V   V  G  E  K                            nsp2      3580       3590       3600       3610       3620       3630UUGUCCGUCC CAGGCAAAAU GGUUGACUGG UUGUCAGACC GGCCUGAGGC UACCUUCAGA  L  S  V   P  G  K   M  V  D  W   L  S  D   R  P  E   A  T  E  R                            nsp2      3640       3650       3660       3670       3680       3690GCUCGGCUGG AUUUAGGCAU CCCAGGUGAU GUGCCCAAAU AUGACAUAAU AUUUGUUAAU  A  R  L   D  L  G   I  P  G  D   V  P  K   Y  D  I   I  F  V  N                            nsp2      3700       3710       3720       3730       3740       3750GUGAGGACCC CAUAUAAAUA CCAUCACUAU CAGCAGUGUG AAGACCAUGC CAUUAAGCUA  V  R  T   P  Y  K   Y  H  H  Y   Q  Q  C   E  D  H   A  I  K  L                            nsp2      3760       3770       3780       3790       3800       3810AGCAUGUUGA CCAAGAAAGC AUGUCUGCAU CUGAAUCCCG GCGGAACCUG UGUCAGCAUA  S  M  L   T  K  K   A  C  L  H   L  N  P   G  G  T   C  V  S  I                            nsp2      3820       3830       3840       3850       3860       3870GGUUAUGGUU ACGCUGACAG GGCCAGCGAA AGCAUCAUUG GUGCUAUAGC GCGGCAGUUC  G  Y  G   Y  A  D   R  A  S  E   S  I  I   G  A  I   A  R  Q  F                            nsp2      3880       3890       3900       3910       3920       3930AAGUUUUCCC GAGUAUGCAA ACCGAAAUCC UCACUUGAGG AGACGGAAGU UCUGUUUGUA  K  F  S   R  V  C   K  P  K  S   S  L  E   E  T  E   V  L  F  V                            nsp2      3940       3950       3960       3970       3980       3990UUCAUUGGGU ACGAUCGCAA GGCCCGUACG CACAAUCCUU ACAAGCUAUC AUCAACCUUG  F  I  G   Y  D  R   K  A  R  T   H  N  P   Y  K  L   S  S  T  L                            nsp2      4000       4010       4020       4030 4032ACCAACAUUU AUACAGGUUC CAGACUCCAC GAAGCCGGAU GU  T  N  I   Y  T  G   S  R  L H    E  A  G   C                            nsp2      4042       4052       4062       4072       4082       4092GCACCCUCAU AUCAUGUGGU GCGAGGGGAU AUUGCCACGG CCACCGAAGG AGUGAUUAUA  A  P  S   Y  H  V   V  R  G  D   I  A  T   A  T  E   G  V  I  I                            nsp3      4102       4112       4122       4132       4142       4152AAUGCUGCUA ACAGCAAAGG ACAACCUGGC GGAGGGGUGU GCGGAGCGCU GUAUAAGAAA  N  A  A   N  S  K   G  Q  P  G   G  G  V   C  G  A   L  Y  K  K                            nsp3      4162       4172       4182       4192       4202       4212UUCCCGGAAA GUUUCGAUUU ACAGCCGAUC GAAGUAGGAA AAGCGCGACU GGUCAAAGGU  F  P  E   S  F  D   L  Q  P  I   E  V  G   K  A  R   L  V  K  G                            nsp3      4222       4232       4242       4252       4262       4272GCAGCUAAAC AUAUCAUUCA UGCCGUAGGA CCAAACUUCA ACAAAGUUUC GGAGGUUGAA  A  A  K   H  I  I   H  A  V  G   P  N  F   N  K  V   S  E  V  E                            nsp3      4282       4292       4302       4312       4322       4332GGUGACAAAC AGUUGGCAGA GGCUUAUGAG UCCAUCGCUA AGAUUGUCAA CGAUAACAAU  G  D  K   Q  L  A   E  A  Y  E   S  I  A   K  I  V   N  D  N  N                            nsp3      4342       4352       4362       4372       4382       4392UACAAGUCAG UAGCGAUUCC ACUGUUGUCC ACCGGCAUCU UUUCCGGGAA CAAAGAUCGA  Y  K  S   V  A  I   P  L  L  S   T  G  I   F  S  G   N  K  D  R                            nsp3      4402       4412       4422       4432       4442       4452CUAACCCAAU CAUUGAACCA UUUGCUGACA GCUUUAGACA CCACUGAUGC AGAUGUAGCC  L  T  Q   S  L  N   H  L  L  T   A  L  D   T  T  D   A  D  V  A                            nsp3      4462       4472       4482       4492       4502       4512AUAUACUGCA GGGACAAGAA AUGGGAAAUG ACUCUCAAGG AAGCAGUGGC UAGGAGAGAA  I  Y  C   R  D  K   K  W  E  M   T  L  K   E  A  V   A  R  R  E                            nsp3      4522       4532       4542       4552       4562       4572GCAGUGGAGG AGAUAUGCAU AUCCGACGAU UCUUCAGUGA CAGAACCUGA UGCAGAGCUG  A  V  E   E  I  C   I  S  D  D   S  S  V   T  E  P   D  A  E  L                            nsp3      4582       4592       4602       4612       4622       4632GUGAGGGUGC AUCCCAAGAG UUCUUUGGCU GGAAGGAAGG GCUACAGCAC AAGCGAUGGC  V  R  V   H  P  K   S  S  L  A   G  R  K   G  Y  S   T  S  D  G                            nsp3      4642       4652       4662       4672       4682       4692AAAACUUUCU CAUAUUUGGA AGGGACCAAG UUUCACCAGG CGGCCAAGGA UAUAGCAGAA  K  T  F   S  Y  L   E  G  T  K   F  H  Q   A  A  K   D  I  A  E                            nsp3      4702       4712       4722       4732       4742       4752AUUAAUGCCA UGUGGCCCGU UGCAACGGAG GCCAAUGAGC AGGUAUGCAU GUAUAUCCUC  I  N  A   M  W  P   V  A  T  E   A  N  E   Q  V  C   M  Y  I  L                            nsp3      4762       4772       4782       4792       4802       4812GGAGAAAGCA UGAGCAGUAU UAGGUCGAAA UGCCCCGUCG AGGAGUCGGA AGCCUCCACA  G  E  S   M  S  S   I  R  S  K   C  P  V   E  E  S   E  A  S  T                            nsp3      4822       4832       4842       4852       4862       4872CCACCUAGCA CGCUGCCUUG CUUGUGCAUC CAUGCCAUGA CUCCAGAAAG AGUACAGCGC  P  P  S   T  L  P   C  L  C  I   H  A  M   T  P  E   R  V  Q  R                            nsp3      4882       4892       4902       4912       4922       4932CUAAAAGCCU CACGUCCAGA ACAAAUUACU GUGUGCUCAU CCUUUCCAUU GCCGAAGUAU  L  K  A   S  R  P   E  Q  I  T   V  C  S   S  F  P   L  P  K  Y                            nsp3      4942       4952       4962       4972       4982       4992AGAAUCACUG GUGUGCAGAA GAUCCAAUGC UCCCAGCCUA UAUUGUUCUC ACCGAAAGUG  R  I  T   G  V  Q   K  I  Q  C   S  Q  P   I  L  F   S  P  K  V                            nsp3      5002       5012       5022       5032       5042       5052CCUGCGUAUA UUCAUCCAAG GAAGUAUCUC GUGGAAACAC CACCGGUAGA CGAGACUCCG  P  A  Y   I  H  P   R  K  Y  L   V  E  T   P  P  V   D  E  T  P                            nsp3      5062       5072       5082       5092       5102       5112GAGCCAUCGG CAGAGAACCA AUCCACAGAG GGGACACCUG AACAACCACC ACUUAUAACC  E  P  S   A  E  N   Q  S  T  E   G  T  P   E  Q  P   P  L  I  T                            nsp3      5122       5132       5142       5152       5162       5172GAGGAUGAGA CCAGGACUAG AACGCCUGAG CCGAUCAUCA UCGAAGAAGA AGAAGAAGAU  E  D  E   T  R  T   R  T  P  E   P  I  I   I  E  E   E  E  E  D                            nsp3      5182       5192       5202       5212       5222       5232AGCAUAAGUU UGCUGUCAGA UGGCCCGACC CACCAGGUGC UGCAAGUCGA GGCAGACAUU  S  I  S   L  L  S   D  G  P  T   H  Q  V   L  Q  V   E  A  D  I                            nsp3      5242       5252       5262       5272       5282       5292CACGGGCCGC CCUCUGUAUC UAGCUCAUCC UGGUCCAUUC CUCAUGCAUC CGACUUUGAU  H  G  P   P  S  V   S  S  S  S   W  S  I   P  H  A   S  D  E  D                            nsp3      5302       5312       5322       5332       5342       5352GUGGACAGUU UAUCCAUACU UGACACCCUG GAGGGAGCUA GCGUGACCAG CGGGGCAACG  V  D  S   L  S  I   L  D  T  L   E  G  A   S  V  T   S  G  A  T                            nsp3      5362       5372       5382       5392       5402       5412UCAGCCGAGA CUAACUCUUA CUUCGCAAAG AGUAUGGAGU UUCUGGCGCG ACCGGUGCCU  S  A  E   T  N  S   Y  F  A  K   S  M  E   F  L  A   R  P  V  P                            nsp3      5422       5432       5442       5452       5462       5472GCGCCUCGAA CAGUAUUCAG GAACCCUCCA CAUCCCGCUC CGCGCACAAG AACACCGUCA  A  P  R   T  V  F   R  N  P  P   H  P  A   P  R  T   R  T  P  S                            nsp3      5482       5492       5502       5512       5522       5532CUUGCACCCA GCAGGGCCUG CUCCAGAACC AGCCUAGUUU CCACCCCGCC AGGCGUGAAU  L  A  P   S  R  A   C  S  R  T   S  L  V   S  T  P   P  G  V  N                            nsp3      5542       5552       5562       5572       5582       5592AGGGUGAUCA CUAGAGAGGA GCUCGAAGCG CUUACCCCGU CACGCACUCC UAGCAGGUCG  R  V  I   T  R  E   E  L  E  A   L  T  P   S  R  T   P  S  R  S                            nsp3      5602       5612       5622       5632       5642       5652GUCUCCAGAA CCAGCCUGGU CUCCAACCCG CCAGGCGUAA AUAGGGUGAU UACAAGAGAG  V  S  R   T  S  L   V  S  N  P   P  G  V   N  R  V   I  T  R  E                            nsp3      5662       5672       5682       5692       5702 5703GAGUUUGAGG CGUUCGUAGC ACAACAACAA UGACGGUUUG AUGCGGGUGC A  E  F  E   A  F  V   A  Q  Q  Q   *  R  F   D  A  G   A                            nsp3      5713       5723       5733       5743       5753       5763UACAUCUUUU CCUCCGACAC CGGUCAAGGG CAUUUACAAC AAAAAUCAGU AAGGCAAACG  Y  I  F   S  S  D   T  G  Q  G   H  L  Q   Q  K  S   V  R  Q  T                            nsp4      5773       5783       5793       5803       5813       5823GUGCUAUCCG AAGUGGUGUU GGAGAGGACC GAAUUGGAGA UUUCGUAUGC CCCGCGCCUC  V  L  S   E  V  V   L  E  R  T   E  L  E   I  S  Y   A  P  R  L                            nsp4      5833       5843       5853       5863       5873       5883GACCAAGAAA AAGAAGAAUU ACUACGCAAG AAAUUACAGU UAAAUCCCAC ACCUGCUAAC  D  Q  E   K  E  E   L  L  R  K   K  L  Q   L  N  P   T  P  A  N                            nsp4      5893       5903       5913       5923       5933       5943AGAAGCAGAU ACCAGUCCAG GAAGGUGGAG AACAUGAAAG CCAUAACAGC UAGACGUAUU  R  S  R   Y  Q  S   R  K  V  E   N  M  K   A  I  T   A  R  R  I                            nsp4      5953       5963       5973       5983       5993       6003CUGCAAGGCC UAGGGCAUUA UUUGAAGGCA GAAGGAAAAG UGGAGUGCUA CCGAACCCUG  L  Q  G   L  G  H   Y  L  K  A   E  G  K   V  E  C   Y  R  T  L                            nsp4      6013       6023       6033       6043       6053       6063CAUCCUGUUC CUUUGUAUUC AUCUAGUGUG AACCGUGCCU UUUCAAGCCC CAAGGUCGCA  H  P  V   P  L  Y   S  S  S  V   N  R  A   F  S  S   P  K  V  A                            nsp4      6073       6083       6093       6103       6113       6123GUGGAAGCCU GUAACGCCAU GUUGAAAGAG AACUUUCCGA CUGUGGCUUC UUACUGUAUU  V  E  A   C  N  A   M  L  K  E   N  F  P   T  V  A   S  Y  C  I                            nsp4      6133       6143       6153       6163       6173       6183AUUCCAGAGU ACGAUGCCUA UUUGGACAUG GUUGACGGAG CUUCAUGCUG CUUAGACACU  I  P  E   Y  D  A   Y  L  D  M   V  D  G   A  S  C   C  L  D  T                            nsp4      6193       6203       6213       6223       6233       6243GCCAGUUUUU GCCCUGCAAA GCUGCGCAGC UUUCCAAAGA AACACUCCUA UUUGGAACCC  A  S  F   C  P  A   K  L  R  S   F  P  K   K  H  S   Y  L  E  P                            nsp4      6253       6263       6273       6283       6293       6303ACAAUACGAU CGGCAGUGCC UUCAGCGAUC CAGAACACGC UCCAGAACGU CCUGGCAGCU  T  I  R   S  A  V   P  S  A  I   Q  N  T   L  Q  N   V  L  A  A                            nsp4      6313       6323       6333       6343       6353       6363GCCACAAAAA GAAAUUGCAA UGUCACGCAA AUGAGAGAAU UGCCCGUAUU GGAUUCGGCG  A  T  K   R  N  C   N  V  T  Q   M  R  E   L  P  V   L  D  S  A                            nsp4      6373       6383       6393       6403       6413       6423GCCUUUAAUG UGGAAUGCUU CAAGAAAUAU GCGUGUAAUA AUGAAUAUUG GGAAACGUUU  A  F  N   V  E  C   F  K  K  Y   A  C  N   N  E  Y   W  E  T  F                            nsp4      6433       6443       6453       6463       6473       6483AAAGAAAACC CCAUCAGGCU UACUGAAGAA AACGUGGUAA AUUACAUUAC CAAAUUAAAA  K  E  N   P  I  R   L  T  E  E   N  V  V   N  Y  I   T  K  L  K                            nsp4      6493       6503       6513       6523       6533       6543GGACCAAAAG CUGCUGCUCU UUUUGCGAAG ACACAUAAUU UGAAUAUGUU GCAGGACAUA  G  P  K   A  A  A   L  F  A  K   T  H  N   L  N  M   L  Q  D  I                            nsp4      6553       6563       6573       6583       6593       6603CCAAUGGACA GGUUUGUAAU GGACUUAAAG AGAGACGUGA AAGUGACUCC AGGAACAAAA  P  M  D   R  F  V   M  D  L  K   R  D  V   K  V  T   P  G  T  K                            nsp4      6613       6623       6633       6643       6653       6663CAUACUGAAG AACGGCCCAA GGUACAGGUG AUCCAGGCUG CCGAUCCGCU AGCAACAGCG  H  T  E   E  R  P   K  V  Q  V   I  Q  A   A  D  P   L  A  T  A                            nsp4      6673       6683       6693       6703       6713       6723UAUCUGUGCG GAAUCCACCG AGAGCUGGUU AGGAGAUUAA AUGCGGUCCU GCUUCCGAAC  Y  L  C   G  I  H   R  E  L  V   R  R  L   N  A  V   L  L  P  N                            nsp4      6733       6743       6753       6763       6773       6783AUUCAUACAC UGUUUGAUAU GUCGGCUGAA GACUUUGACG CUAUUAUAGC CGAGCACUUC  I  H  T   L  E  D   M  S  A  E   D  F  D   A  I  I   A  E  H  F                            nsp4      6793       6803       6813       6823       6833       6843CAGCCUGGGG AUUGUGUUCU GGAAACUGAC AUCGCGUCGU UUGAUAAAAG UGAGGACGAC  Q  P  G   D  C  V   L  E  T  D   I  A  S   F  D  K   S  E  D  D                            nsp4      6853       6863       6873       6883       6893       6903GCCAUGGCUC UGACCGCGUU AAUGAUUCUG GAAGACUUAG GUGUGGACGC AGAGCUGUUG  A  M  A   L  T  A   L  M  I  L   E  D  L   G  V  D   A  E  L  L                            nsp4      6913       6923       6933       6943       6953       6963ACGCUGAUUG AGGCGGCUUU CGGCGAAAUU UCAUCAAUAC AUUUGCCCAC UAAAACUAAA  T  L  I   E  A  A   F  G  E  I   S  S  I   H  L  P   T  K  T  K                            nsp4      6973       6983       6993       7003       7013       7023UUUAAAUUCG GAGCCAUGAU GAAAUCUGGA AUGUUCCUCA CACUGUUUGU GAACACAGUC  F  K  F   G  A  M   M  K  S  G   M  F  L   T  L  F   V  N  T  V                            nsp4      7033       7043       7053       7063       7073       7083AUUAACAUUG UAAUCGCAAG CAGAGUGUUG AGAGAACGGC UAACCGGAUC ACCAUGUGCA  I  N  I   V  I  A   S  R  V  L   R  E  R   L  T  G   S  P  C  A                            nsp4      7093       7103       7113       7123       7133       7143GCAUUCAUUG GAGAUGACAA UAUCGUGAAA GGAGUCAAAU CGGACAAAUU AAUGGCAGAC  A  F  I   G  D  D   N  I  V  K   G  V  K   S  D  K   L  M  A  D                            nsp4      7153       7163       7173       7183       7193       7203AGGUGCGCCA CCUGGUUGAA UAUGGAAGUC AAGAUUAUAG AUGCUGUGGU GGGCGAGAAA  R  C  A   T  W  L   N  M  E  V   K  I  I   D  A  V   V  G  E  K                            nsp4      7213       7223       7233       7243       7253       7263GCGCCUUAUU UCUGUGGAGG GUUUAUUUUG UGUGACUCCG UGACCGGCAC AGCGUGCCGU  A  P  Y   F  C  G   G  E  I  L   C  D  S   V  T  G   T  A  C  R                            nsp4      7273       7283       7293       7303       7313       7323GUGGCAGACC CCCUAAAAAG GCUGUUUAAG CUAGGCAAAC CUCUGGCAGC AGACGAUGAA  V  A  D   P  L  K   R  L  F  K   L  G  K   P  L  A   A  D  D  E                            nsp4      7333       7343       7353       7363       7373       7383CAUGAUGAUG ACAGGAGAAG GGCAUUGCAU GAGGAGUCAA CACGCUGGAA CCGAGUGGGU  H  D  D   D  R  R   R  A  L  H   E  E  S   T  R  W   N  R  V  G                            nsp4      7393       7403 7      413       7423       7433       7443AUUCUUUCAG AGCUGUGCAA GGCAGUAGAA UCAAGGUAUG AAACCGUAGG AACUUCCAUC  I  L  S   E  L  C   K  A  V  E   S  R  Y   E  T  V   G  T  S  I                            nsp4      7453       7463       7473       7483       7493       7503AUAGUUAUGG CCAUGACUAC UCUAGCUAGC AGUGUUAAAU CAUUCAGCUA CCUGAGAGGG  I  V  M   A  M  T   T  L  A  S   S  V  K   S  F  S   Y  L  R  G                            nsp4       7513       7523 7527GCCCCUAUAA CUCUCUACGG CUAA   A  P  I   T  L  Y   G  *                            nsp4      7537       7547       7557       7567 7568CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAGACUAG U                          virUTR      7578       7588       7598       7608       7618       7628AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGGU GAGAUUUCCA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   V  R  F  P                          RBD (S protein)      7638       7648       7658       7668       7678       7688AAUAUUACAA AUCUGUGUCC AUUUGGAGAA GUGUUUAAUG CAACAAGAUU UGCAUCUGUG  N  I  T   N  L  C   P  F  G  E   V  F  N   A  T  R   F  A  S  V                          RBD (S protein)      7698       7708       7718       7728       7738       7748UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU  Y  A  W   N  R  K   R  I  S  N   C  V  A   D  Y  S   V  L  Y  N                          RBD (S protein)      7758       7768       7778       7788       7798       7808AGUGCUUCUU UUUCCACAUU UAAAUGUUAU GGAGUGUCUC CAACAAAAUU AAAUGAUUUA  S  A  S   F  S  T   F  K  C  Y   G  V  S   P  T  K   L  N  D  L                          RBD (S protein)      7818       7828       7838       7848       7858       7868UGUUUUACAA AUGUGUAUGC UGAUUCUUUU GUGAUCAGAG GUGAUGAAGU GAGACAGAUU  C  F  T   N  V  Y   A  D  S  F   V  I  R   G  D  E   V  R  Q  I                          RBD (S protein)      7878       7888       7898       7908       7918       7928GCCCCCGGAC AGACAGGAAA AAUUGCUGAU UACAAUUACA AACUGCCUGA UGAUUUUACA  A  P  G   Q  T  G   K  I  A  D   Y  N  Y   K  L  P   D  D  F  T                          RBD (S protein)      7938       7948       7958       7968       7978       7988GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU UUAGAUUCUA AAGUGGGAGG AAAUUACAAU  G  C  V   I  A  W   N  S  N  N   L  D  S   K  V  G   G  N  Y  N                          RBD (S protein)      7998       8008       8018       8028       8038       8048UAUCUGUACA GACUGUUUAG AAAAUCAAAU CUGAAACCUU UUGAAAGAGA UAUUUCAACA  Y  L  Y   R  L  F   R  K  S  N   L  K  P   F  E  R   D  I  S  T                          RBD (S protein)      8058       8068       8078       8088       8098       8108GAAAUUUAUC AGGCUGGAUC AACACCUUGU AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU  E  I  Y   Q  A  G   S  T  P  C   N  G  V   E  G  F   N  C  Y  F                          RBD (S protein)      8118       8128       8138       8148       8158       8168CCAUUACAGA GCUAUGGAUU UCAGCCAACC AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG  P  L  Q   S  Y  G   E  Q  P  T   N  G  V   G  Y  Q   P  Y  R  V                          RBD (S protein)      8178       8188       8198       8208       8218   8222GUGGUGCUGU CUUUUGAACU GCUGCAUGCA CCUGCAACAG UGUGUGGACC UAAA  V  V  L   S  F  E   L  L  H  A   P  A  T   V  C  G   P  K                          RBD (S protein)      8232       8242       8249 GGCUCCCCCG GCUCCGGCUC CGGAUCU  G  S  P   G  S  G   S  G  S                           GS linker      8259       8269       8279       8289       8299       8309GGUUAUAUUC CUGAAGCUCC AAGAGAUGGG CAAGCUUACG UUCGUAAAGA UGGCGAAUGG  G  Y  I   P  E  A   P  R  D  G   Q  A  Y   V  R  K   D  G  E  W                          fibritin      8319       8329       8339       8349       8359       8369GUAUUACUUU CUACCUUUUU AGGCCGGUCC CUGGAGGUGC UGUUCCAGGG CCCCGGCUGA  V  L  L   S  T  F   L  G  R  S   L  E  V   L  F  Q   G  P  G  *                          fibritin 8372 UGA   *                          fibritin      8382       8392       8402       8412       8422       8432CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element      8442       8452       8462       8472       8482       8492AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element      8502       8512       8522       8532       8542       8552UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element      8562       8572       8582       8592       8602       8612CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element      8622       8632       8642       8652       8662       8672GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCGCGGCC GCAUGAAUAC                          FI element      8682       8692       8702       8712       8722       8732AGCAGCAAUU GGCAAGCUGC UUACAUAGAA CUCGCGGCGA UUGGCAUGCC GCCUUAAAAU                          FI element      8742       8752       8762       8772       8782  8786UUUUAUUUUA UUUUUUCUUU UCUUUUCCGA AUCGGAUUUU GUUUUUAAUA UUUC                          FI element      8796       8806       8816       8826       8836       8846AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                          Poly(A)      8856       8866       8876       8886       8896AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          Poly(A)

Nucleotide Sequence of RBS004.4 (SEQ ID NO: 27; SEQ ID NO: 28)Nucleotide sequence is shown with individual sequence elements as indicated in boldletters. In addition, the sequence of the translated protein is shown in italic lettersbelow the coding nucleotide sequence (* = stop codon).        10         20         30         40    45GAUGGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA                           5′UTR        55         65         75         85         95        105AUGGAGAAAG UUCACGUUGA CAUCGAGGAA GACAGCCCAU UCCUCAGAGC UUUGCAGCGG  M  E  K   V  H  V   D  I  E  E   D  S  P   F  L  R   A  L  Q  R                            nsp1       115        125        135        145        155        165AGCUUCCCGC AGUUUGAGGU AGAAGCCAAG CAGGUCACUG AUAAUGACCA UGCUAAUGCC  S  F  P   Q  F  E   V  E  A  K   Q  V  T   D  N  D   H  A  N  A                            nsp1       175        185        195        205        215        225AGAGCGUUUU CGCAUCUGGC UUCAAAACUG AUCGAAACGG AGGUGGACCC AUCCGACACG  R  A  F   S  H  L   A  S  K  L  I  E  T    E  V  D   P  S  D  T                            nsp1       235        245        255        265        275        285AUCCUUGACA UUGGAAGUGC GCCCGCCCGC AGAAUGUAUU CUAAGCACAA GUAUCAUUGU  I  L  D   I  G  S   A  P  A  R  R  M  Y    S  K  H   K  Y  H  C                            nsp1       295        305        315        325        335        345AUCUGUCCGA UGAGAUGUGC GGAAGAUCCG GACAGAUUGU AUAAGUAUGC AACUAAGCUG I  C  P    M  R  C   A  E  D  P   D  R  L   Y  K  Y   A  T  K  L                            nsp1       355        365        375        385        395        405AAGAAAAACU GUAAGGAAAU AACUGAUAAG GAAUUGGACA AGAAAAUGAA GGAGCUCGCC  K  K  N   C  K  E   I  T  D  K   E  L  D   K  K  M   K  E  L  A                            nsp1       415        425        435        445        455        465GCCGUCAUGA GCGACCCUGA CCUGGAAACU GAGACUAUGU GCCUCCACGA CGACGAGUCG  A  V  M   S  D  P   D  L  E  T   E  T  M   C  L  H   D  D  E  S                            nsp1       475        485        495        505        515        525UGUCGCUACG AAGGGCAAGU CGCUGUUUAC CAGGAUGUAU ACGCGGUUGA CGGACCGACA  C  R  Y   E  G  Q   V  A  V  Y   Q  D  V   Y  A  V   D  G  P  T                            nsp1       535        545        555        565        575        585AGUCUCUAUC ACCAAGCCAA UAAGGGAGUU AGAGUCGCCU ACUGGAUAGG CUUUGACACC  S  L  Y   H  Q  A   N  K  G  V   R  V  A   Y  W  I   G  F  D  T                            nsp1       595        605        615        625        635        645ACCCCUUUUA UGUUUAAGAA CUUGGCUGGA GCAUAUCCAU CAUACUCUAC CAACUGGGCC  T  P  F   M  F  K   N  L  A  G   A  Y  P   S  Y  S   T  N  W  A                            nsp1       655        665        675        685        695        705GACGAAACCG UGUUAACGGC UCGUAACAUA GGCCUAUGCA GCUCUGACGU UAUGGAGCGG  D  E  T   V  L  T   A  R  N  I   G  L  C   S  S  D   V  M  E  R                            nsp1       715        725        735        745        755        765UCACGUAGAG GGAUGUCCAU UCUUAGAAAG AAGUAUUUGA AACCAUCCAA CAAUGUUCUA  S  R  R   G  M  S   I  L  R  K   K  Y  L   K  P  S   N  N  V  L                            nsp1       775        785        795        805        815        825UUCUCUGUUG GCUCGACCAU CUACCACGAA AAGAGGGACU UACUGAGGAG CUGGCACCUG  F  S  V   G  S  T   I  Y  H  E   K  R  D   L  L  R   S  W  H  L                            nsp1       835        845        855        865        875        885CCGUCUGUAU UUCACUUACG UGGCAAGCAA AAUUACACAU GUCGGUGUGA GACUAUAGUU  P  S  V   F  H  L   R  G  K  Q   N  Y  T   C  R  C   E  T  I  V                            nsp1       895        905        915        925        935        945AGUUGCGACG GGUACGUCGU UAAAAGAAUA GCUAUCAGUC CAGGCCUGUA UGGGAAGCCU  S  C  D   G  Y  V   V  K  R  I   A  I  S   P  G  L   Y  G  K  P                            nsp1       955        965        975        985        995       1005UCAGGCUAUG CUGCUACGAU GCACCGCGAG GGAUUCUUGU GCUGCAAAGU GACAGACACA  S  G  Y   A  A  T   M  H  R  E   G  E  L   C  C  K   V  T  D  T                            nsp1      1015       1025       1035       1045       1055       1065UUGAACGGGG AGAGGGUCUC UUUUCCCGUG UGCACGUAUG UGCCAGCUAC AUUGUGUGAC  L  N  G   E  R  V   S  F  P  V   C  T  Y   V  P  A   T  L  C  D                            nsp1      1075       1085       1095       1105       1115       1125CAAAUGACUG GCAUACUGGC AACAGAUGUC AGUGCGGACG ACGCGCAAAA ACUGCUGGUU  Q  M  T   G  I  L   A  T  D  V   S  A  D   D  A  Q   K  L  L  V                            nsp1      1135       1145       1155       1165       1175       1185GGGCUCAACC AGCGUAUAGU CGUCAACGGU CGCACCCAGA GAAACACCAA UACCAUGAAA  G  L  N   Q  R  I   V  V  N  G   R  T  Q   R  N  T   N  T  M  K                            nsp1      1198       1205       1215       1225       1235       1245AAUUACCUUU UGCCCGUAGU GGCCCAGGCA UUUGCUAGGU GGGCAAAGGA AUAUAAGGAA  N  Y  L   L  P  V   V  A  Q  A   F  A  R   W  A  K   E  Y  K  E                            nsp1      1255       1265       1275       1285       1295       1305GAUCAAGAAG AUGAAAGGCC ACUAGGACUA CGAGAUAGAC AGUUAGUCAU GGGGUGUUGU  D  Q  E   D  E  R   P  L  G  L   R  D  R   Q  L  V   M  G  C  C                            nsp1      1315       1325       1335       1345       1355       1365UGGGCUUUUA GAAGGCACAA GAUAACAUCU AUUUAUAAGC GCCCGGAUAC CCAAACCAUC  W  A  F   R  R  H   K  I  T  S   I  Y  K   R  P  D  T  Q  T  I                            nsp1      1375       1385       1395       1405       1415       1425AUCAAAGUGA ACAGCGAUUU CCACUCAUUC GUGCUGCCCA GGAUAGGCAG UAACACAUUG  I  K  V   N  S  D   F  H  S  F   V  L  P   R  I  G   S  N  T  L                            nsp1      1435       1445       1455       1465       1475       1485GAGAUCGGGC UGAGAACAAG AAUCAGGAAA AUGUUAGAGG AGCACAAGGA GCCGUCACCU  E  I  G   L  R  T   R  I  R  K   M  L  E   E  H  K   E  P  S  P                            nsp1      1495       1505       1515       1525       1535       1545CUCAUUACCG CCGAGGACGU ACAAGAAGCU AAGUGCGCAG CCGAUGAGGC UAAGGAGGUG  L  I  T   A  E  D   V  Q  E  A   K  C  A   A  D  E   A  K  E  V                            nsp1      1555       1565       1575       1585       1595       1605CGUGAAGCCG AGGAGUUGCG CGCAGCUCUA CCACCUUUGG CAGCUGAUGU UGAGGAGCCC  R  E  A   E  E  L   R  A  A  L   P  P  L   A  A  D   V  E  E  P                            nsp1      1615       1625       1635       1645       1650ACUCUGGAAG CCGAUGUCGA CUUGAUGUUA CAAGAGGCUG GGGCC  T  L  E   A  D  V   D  L  M  L   Q  E  A   G  A                            nsp1      1660       1670       1680       1690       1700       1710GGCUCAGUGG AGACACCUCG UGGCUUGAUA AAGGUUACCA GCUACGCUGG CGAGGACAAG  G  S  V   E  T  P   R  G  L  I   K  V  T   S  Y  A   G  E  D  K                            nsp2      1720       1730       1740       1750       1760       1770AUCGGCUCUU ACGCUGUGCU UUCUCCGCAG GCUGUACUCA AGAGUGAAAA AUUAUCUUGC  I  G  S   Y  A  V   L  S  P  Q   A  V  L   K  S  E   K  L  S  C                            nsp2      1780       1790       1800       1810       1820       1830AUCCACCCUC UCGCUGAACA AGUCAUAGUG AUAACACACU CUGGCCGAAA AGGGCGUUAU  I  H  P   L  A  E   Q  V  I  V   I  T  H   S  G  R   K  G  R  Y                            nsp2      1840       1850       1860       1870       1880       1890GCCGUGGAAC CAUACCAUGG UAAAGUAGUG GUGCCAGAGG GACAUGCAAU ACCCGUCCAG  A  V  E   P  Y  H   G  K  V  V   V  P  E   G  H  A   I  P  V  Q                            nsp2      1900       1910       1920       1930       1940       1950GACUUUCAAG CUCUGAGUGA AAGUGCCACC AUUGUGUACA ACGAACGUGA GUUCGUAAAC  D  F  Q   A  L  S   E  S  A  T   I  V  Y   N  E  R   E  F  V  N                            nsp2      1960       1970       1980       1990       2000       2010AGGUACCUGC ACCAUAUUGC CACACAUGGA GGAGCGCUGA ACACUGAUGA AGAAUAUUAC  R  Y  L   H  H  I   A  T  H  G   G  A  L   N  T  D   E  E  Y  Y                            nsp2      2020       2030       2040       2050       2060       2070AAAACUGUCA AGCCCAGCGA GCACGACGGC GAAUACCUGU ACGACAUCGA CAGGAAACAG  K  T  V   K  P  S   E  H  D  G   E  Y  L   Y  D  I   D  R  K  Q                            nsp2      2080       2090       2100       2110       2120       2130UGCGUCAAGA AAGAGCUAGU CACUGGGCUA GGGCUCACAG GCGAGCUGGU CGAUCCUCCC  C  V  K   K  E  L   V  T  G  L   G  L  T   G  E  L   V  D  P  P                            nsp2      2140       2150       2160       2170       2180       2190UUCCAUGAAU UCGCCUACGA GAGUCUGAGA ACACGACCAG CCGCUCCUUA CCAAGUACCA  F  H  E   F  A  Y   E  S  L  R   T  R  P   A  A  P   Y  Q  V  P                            nsp2      2200       2210       2220       2230       2240       2250ACCAUAGGGG UGUAUGGCGU GCCAGGAUCA GGCAAGUCUG GCAUCAUUAA AAGCGCAGUC  T  I  G   V  Y  G   V  P  G  S   G  K  S   G  I  I   K  S  A  V                            nsp2      2260       2270       2280       2290       2300       2310ACCAAAAAAG AUCUAGUGGU GAGCGCCAAG AAAGAAAACU GUGCAGAAAU UAUAAGGGAC  T  K  K   D  L  V   V  S  A  K   K  E  N   C  A  E   I  I  R  D                            nsp2      2320       2330       2340       2350       2360       2370GUCAAGAAAA UGAAAGGGCU GGACGUCAAU GCCAGAACUG UGGACUCAGU GCUCUUGAAU  V  K  K   M  K  G   L  D  V  N   A  R  T   V  D  S   V  L  L  N                            nsp2      2380       2390       2400       2410       2420       2430GGAUGCAAAC ACCCCGUAGA GACCCUGUAU AUUGACGAGG CUUUUGCUUG UCAUGCAGGU  G  C  K   H  P  V   E  T  L  Y   I  D  E   A  F  A   C  H  A  G                            nsp2      2440       2450       2460       2470       2480       2490ACUCUCAGAG CGCUCAUAGC CAUUAUAAGA CCUAAAAAGG CAGUGCUCUG CGGAGAUCCC  T  L  R   A  L  I   A  I  I  R   P  K  K   A  V  L   C  G  D  P                            nsp2      2500       2510       2520       2530       2540       2550AAACAGUGCG GUUUUUUUAA CAUGAUGUGC CUGAAAGUGC AUUUUAACCA CGAGAUUUGC  K  Q  C   G  E  E   N  M  M  C   L  K  V   H  F  N   H  E  I  C                            nsp2      2560       2570       2580       2590       2600       2610ACACAAGUCU UCCACAAAAG CAUCUCUCGC CGUUGCACUA AAUCUGUGAC UUCGGUCGUC  T  Q  V   F  H  K   S  I  S  R   R  C  T   K  S  V   T  S  V  V                            nsp2      2620       2630       2640       2650       2660       2670UCAACCUUGU UUUACGACAA AAAAAUGAGA ACGACGAAUC CGAAAGAGAC UAAGAUUGUG  S  T  L   F  Y  D   K  K  M  R   T  T  N   P  K  E   T  K  I  V                            nsp2      2680       2690       2700       2710       2720       2730AUUGACACUA CCGGCAGUAC CAAACCUAAG CAGGACGAUC UCAUUCUCAC UUGUUUCAGA  I  D  T   T  G  S   T  K  P  K   Q  D  D   L  I  L   T  C  F  R                            nsp2      2740       2750       2760       2770       2780       2790GGGUGGGUGA AGCAGUUGCA AAUAGAUUAC AAAGGCAACG AAAUAAUGAC GGCAGCUGCC  G  W  V   K  Q  L   Q  I  D  Y   K  G  N   E  I  M   T  A  A  A                            nsp2      2800       2810       2820       2830       2840       2850UCUCAAGGGC UGACCCGUAA AGGUGUGUAU GCCGUUCGGU ACAAGGUGAA UGAAAAUCCU  S  Q  G   L  T  R   K  G  V  Y   A  V  R   Y  K  V   N  E  N  P                            nsp2      2860       2870       2880       2890       2900       2910CUGUACGCAC CCACCUCAGA ACAUGUGAAC GUCCUACUGA CCCGCACGGA GGACCGCAUC  L  Y  A   P  T  S   E  H  V  N   V  L  L   T  R  T   E  D  R  I                            nsp2      2920       2930       2940       2950       2960       2970GUGUGGAAAA CACUAGCCGG CGACCCAUGG AUAAAAACAC UGACUGCCAA GUACCCUGGG  V  W  K   T  L  A   G  D  P  W   I  K  T   L  T  A   K  Y  P  G                            nsp2      2980       2990       3000       3010       3020       3030AAUUUCACUG CCACGAUAGA GGAGUGGCAA GCAGAGCAUG AUGCCAUCAU GAGGCACAUC  N  F  T   A  T  I   E  E  W  Q   A  E  H   D  A  I   M  R  H  I                            nsp2      3040       3050       3060       3070       3080       3090UUGGAGAGAC CGGACCCUAC CGACGUCUUC CAGAAUAAGG CAAACGUGUG UUGGGCCAAG  L  E  R   P  D  P   T  D  V  F   Q  N  K   A  N  V   C  W  A  K                            nsp2      3100       3110       3120       3130       3140       3150GCUUUAGUGC CGGUGCUGAA GACCGCUGGC AUAGACAUGA CCACUGAACA AUGGAACACU  A  L  V   P  V  L   K  T  A  G   I  D  M   T  T  E   Q  W  N  T                            nsp2      3160       3170       3180       3190       3200       3210GUGGAUUAUU UUGAAACGGA CAAAGCUCAC UCAGCAGAGA UAGUAUUGAA CCAACUAUGC  V  D  Y   F  E  T   D  K  A  H   S  A  E   I  V  L   N  Q  L  C                            nsp2      3220       3230       3240       3250       3260       3270GUGAGGUUCU UUGGACUCGA UCUGGACUCC GGUCUAUUUU CUGCACCCAC UGUUCCGUUA  V  R  F   F  G  L   D  L  D  S   G  L  F   S  A  P   T  V  P  L                            nsp2      3280       3290       3300       3310       3320       3330UCCAUUAGGA AUAAUCACUG GGAUAACUCC CCGUCGCCUA ACAUGUACGG GCUGAAUAAA  S  I  R   N  N  H   W  D  N  S   P  S  P   N  M  Y   G  L  N  K                            nsp2      3340       3350       3360       3370       3380       3390GAAGUGGUCC GUCAGCUCUC UCGCAGGUAC CCACAACUGC CUCGGGCAGU UGCCACUGGU  E  V  V   R  Q  L   S  R  R  Y   P  Q  L   P  R  A   V  A  T  G                            nsp2      3400       3410       3420       3430       3440       3450AGAGUCUAUG ACAUGAACAC UGGUACACUG CGCAAUUAUG AUCCGCGCAU AAACCUAGUA  R  V  Y   D  M  N   T  G  T  L   R  N  Y   D  P  R   I  N  L  V                            nsp2      3460       3470       3480       3490       3500       3510CCUGUAAACA GAAGACUGCC UCAUGCUUUA GUCCUCCACC AUAAUGAACA CCCACAGAGU  P  V  N   R  R  L   P  H  A  L   V  L  H   H  N  E   H  P  Q  S                            nsp2      3520       3530       3540       3550       3560       3570GACUUUUCUU CAUUCGUCAG CAAAUUGAAG GGCAGAACUG UCCUGGUGGU CGGGGAAAAG  D  F  S   S  F  V   S  K  L  K   G  R  T   V  L  V   V  G  E  K                            nsp2      3580       3590       3600       3610       3620       3630UUGUCCGUCC CAGGCAAAAU GGUUGACUGG UUGUCAGACC GGCCUGAGGC UACCUUCAGA  L  S  V   P  G  K   M  V  D  W   L  S  D   R  P  E   A  T  E  R                            nsp2      3640       3650       3660       3670       3680       3690GCUCGGCUGG AUUUAGGCAU CCCAGGUGAU GUGCCCAAAU AUGACAUAAU AUUUGUUAAU  A  R  L   D  L  G   I  P  G  D   V  P  K   Y  D  I   I  F  V  N                            nsp2      3700       3710       3720       3730       3740       3750GUGAGGACCC CAUAUAAAUA CCAUCACUAU CAGCAGUGUG AAGACCAUGC CAUUAAGCUA  V  R  T   P  Y  K   Y  H  H  Y   Q  Q  C   E  D  H   A  I  K  L                            nsp2      3760       3770       3780       3790       3800       3810AGCAUGUUGA CCAAGAAAGC AUGUCUGCAU CUGAAUCCCG GCGGAACCUG UGUCAGCAUA  S  M  L   T  K  K   A  C  L  H   L  N  P   G  G  T   C  V  S  I                            nsp2      3820       3830       3840       3850       3860       3870GGUUAUGGUU ACGCUGACAG GGCCAGCGAA AGCAUCAUUG GUGCUAUAGC GCGGCAGUUC  G  Y  G   Y  A  D   R  A  S  E   S  I  I   G  A  I   A  R  Q  F                            nsp2      3880       3890       3900       3910       3920       3930AAGUUUUCCC GAGUAUGCAA ACCGAAAUCC UCACUUGAGG AGACGGAAGU UCUGUUUGUA  K  F  S   R  V  C   K  P  K  S   S  L  E   E  T  E   V  L  F  V                            nsp2      3940       3950       3960       3970       3980       3990UUCAUUGGGU ACGAUCGCAA GGCCCGUACG CACAAUCCUU ACAAGCUAUC AUCAACCUUG  F  I  G   Y  D  R   K  A  R  T   H  N  P   Y  K  L   S  S  T  L                            nsp2      4000       4010       4020       4030 4032ACCAACAUUU AUACAGGUUC CAGACUCCAC GAAGCCGGAU GU  T  N  I   Y  T  G   S  R  L H    E  A  G   C                            nsp2      4042       4052       4062       4072       4082       4092GCACCCUCAU AUCAUGUGGU GCGAGGGGAU AUUGCCACGG CCACCGAAGG AGUGAUUAUA  A  P  S   Y  H  V   V  R  G  D   I  A  T   A  T  E   G  V  I  I                            nsp3      4102       4112       4122       4132       4142       4152AAUGCUGCUA ACAGCAAAGG ACAACCUGGC GGAGGGGUGU GCGGAGCGCU GUAUAAGAAA  N  A  A   N  S  K   G  Q  P  G   G  G  V   C  G  A   L  Y  K  K                            nsp3      4162       4172       4182       4192       4202       4212UUCCCGGAAA GUUUCGAUUU ACAGCCGAUC GAAGUAGGAA AAGCGCGACU GGUCAAAGGU  F  P  E   S  F  D   L  Q  P  I   E  V  G   K  A  R   L  V  K  G                            nsp3      4222       4232       4242       4252       4262       4272GCAGCUAAAC AUAUCAUUCA UGCCGUAGGA CCAAACUUCA ACAAAGUUUC GGAGGUUGAA  A  A  K   H  I  I   H  A  V  G   P  N  F   N  K  V   S  E  V  E                            nsp3      4282       4292       4302       4312       4322       4332GGUGACAAAC AGUUGGCAGA GGCUUAUGAG UCCAUCGCUA AGAUUGUCAA CGAUAACAAU  G  D  K   Q  L  A   E  A  Y  E   S  I  A   K  I  V   N  D  N  N                            nsp3      4342       4352       4362       4372       4382       4392UACAAGUCAG UAGCGAUUCC ACUGUUGUCC ACCGGCAUCU UUUCCGGGAA CAAAGAUCGA  Y  K  S   V  A  I   P  L  L  S   T  G  I   F  S  G   N  K  D  R                            nsp3      4402       4412       4422       4432       4442       4452CUAACCCAAU CAUUGAACCA UUUGCUGACA GCUUUAGACA CCACUGAUGC AGAUGUAGCC  L  T  Q   S  L  N   H  L  L  T   A  L  D   T  T  D   A  D  V  A                            nsp3      4462       4472       4482       4492       4502       4512AUAUACUGCA GGGACAAGAA AUGGGAAAUG ACUCUCAAGG AAGCAGUGGC UAGGAGAGAA  I  Y  C   R  D  K   K  W  E  M   T  L  K   E  A  V   A  R  R  E                            nsp3      4522       4532       4542       4552       4562       4572GCAGUGGAGG AGAUAUGCAU AUCCGACGAU UCUUCAGUGA CAGAACCUGA UGCAGAGCUG  A  V  E   E  I  C   I  S  D  D   S  S  V   T  E  P   D  A  E  L                            nsp3      4582       4592       4602       4612       4622       4632GUGAGGGUGC AUCCCAAGAG UUCUUUGGCU GGAAGGAAGG GCUACAGCAC AAGCGAUGGC  V  R  V   H  P  K   S  S  L  A   G  R  K   G  Y  S   T  S  D  G                            nsp3      4642       4652       4662       4672       4682       4692AAAACUUUCU CAUAUUUGGA AGGGACCAAG UUUCACCAGG CGGCCAAGGA UAUAGCAGAA  K  T  F   S  Y  L   E  G  T  K   F  H  Q   A  A  K   D  I  A  E                            nsp3      4702       4712       4722       4732       4742       4752AUUAAUGCCA UGUGGCCCGU UGCAACGGAG GCCAAUGAGC AGGUAUGCAU GUAUAUCCUC  I  N  A   M  W  P   V  A  T  E   A  N  E   Q  V  C   M  Y  I  L                            nsp3      4762       4772       4782       4792       4802       4812GGAGAAAGCA UGAGCAGUAU UAGGUCGAAA UGCCCCGUCG AGGAGUCGGA AGCCUCCACA  G  E  S   M  S  S   I  R  S  K   C  P  V   E  E  S   E  A  S  T                            nsp3      4822       4832       4842       4852       4862       4872CCACCUAGCA CGCUGCCUUG CUUGUGCAUC CAUGCCAUGA CUCCAGAAAG AGUACAGCGC  P  P  S   T  L  P   C  L  C  I   H  A  M   T  P  E   R  V  Q  R                            nsp3      4882       4892       4902       4912       4922       4932CUAAAAGCCU CACGUCCAGA ACAAAUUACU GUGUGCUCAU CCUUUCCAUU GCCGAAGUAU  L  K  A   S  R  P   E  Q  I  T   V  C  S   S  F  P   L  P  K  Y                            nsp3      4942       4952       4962       4972       4982       4992AGAAUCACUG GUGUGCAGAA GAUCCAAUGC UCCCAGCCUA UAUUGUUCUC ACCGAAAGUG  R  I  T   G  V  Q   K  I  Q  C   S  Q  P   I  L  F   S  P  K  V                            nsp3      5002       5012       5022       5032       5042       5052CCUGCGUAUA UUCAUCCAAG GAAGUAUCUC GUGGAAACAC CACCGGUAGA CGAGACUCCG  P  A  Y   I  H  P   R  K  Y  L   V  E  T   P  P  V   D  E  T  P                            nsp3      5062       5072       5082       5092       5102       5112GAGCCAUCGG CAGAGAACCA AUCCACAGAG GGGACACCUG AACAACCACC ACUUAUAACC  E  P  S   A  E  N   Q  S  T  E   G  T  P   E  Q  P   P  L  I  T                            nsp3      5122       5132       5142       5152       5162       5172GAGGAUGAGA CCAGGACUAG AACGCCUGAG CCGAUCAUCA UCGAAGAAGA AGAAGAAGAU  E  D  E   T  R  T   R  T  P  E   P  I  I   I  E  E   E  E  E  D                            nsp3      5182       5192       5202       5212       5222       5232AGCAUAAGUU UGCUGUCAGA UGGCCCGACC CACCAGGUGC UGCAAGUCGA GGCAGACAUU  S  I  S   L  L  S   D  G  P  T   H  Q  V   L  Q  V   E  A  D  I                            nsp3      5242       5252       5262       5272       5282       5292CACGGGCCGC CCUCUGUAUC UAGCUCAUCC UGGUCCAUUC CUCAUGCAUC CGACUUUGAU  H  G  P   P  S  V   S  S  S  S   W  S  I   P  H  A   S  D  E  D                            nsp3      5302       5312       5322       5332       5342       5352GUGGACAGUU UAUCCAUACU UGACACCCUG GAGGGAGCUA GCGUGACCAG CGGGGCAACG  V  D  S   L  S  I   L  D  T  L   E  G  A   S  V  T   S  G  A  T                            nsp3      5362       5372       5382       5392       5402       5412UCAGCCGAGA CUAACUCUUA CUUCGCAAAG AGUAUGGAGU UUCUGGCGCG ACCGGUGCCU  S  A  E   T  N  S   Y  F  A  K   S  M  E   F  L  A   R  P  V  P                            nsp3      5422       5432       5442       5452       5462       5472GCGCCUCGAA CAGUAUUCAG GAACCCUCCA CAUCCCGCUC CGCGCACAAG AACACCGUCA  A  P  R   T  V  F   R  N  P  P   H  P  A   P  R  T   R  T  P  S                            nsp3      5482       5492       5502       5512       5522       5532CUUGCACCCA GCAGGGCCUG CUCCAGAACC AGCCUAGUUU CCACCCCGCC AGGCGUGAAU  L  A  P   S  R  A   C  S  R  T   S  L  V   S  T  P   P  G  V  N                            nsp3      5542       5552       5562       5572       5582       5592AGGGUGAUCA CUAGAGAGGA GCUCGAAGCG CUUACCCCGU CACGCACUCC UAGCAGGUCG  R  V  I   T  R  E   E  L  E  A   L  T  P   S  R  T   P  S  R  S                            nsp3      5602       5612       5622       5632       5642       5652GUCUCCAGAA CCAGCCUGGU CUCCAACCCG CCAGGCGUAA AUAGGGUGAU UACAAGAGAG  V  S  R   T  S  L   V  S  N  P   P  G  V   N  R  V   I  T  R  E                            nsp3      5662       5672       5682       5692       5702 5703GAGUUUGAGG CGUUCGUAGC ACAACAACAA UGACGGUUUG AUGCGGGUGC A  E  F  E   A  F  V   A  Q  Q  Q   *  R  F   D  A  G   A                            nsp3      5713       5723       5733       5743       5753       5763UACAUCUUUU CCUCCGACAC CGGUCAAGGG CAUUUACAAC AAAAAUCAGU AAGGCAAACG  Y  I  F   S  S  D   T  G  Q  G   H  L  Q   Q  K  S   V  R  Q  T                            nsp4      5773       5783       5793       5803       5813       5823GUGCUAUCCG AAGUGGUGUU GGAGAGGACC GAAUUGGAGA UUUCGUAUGC CCCGCGCCUC  V  L  S   E  V  V   L  E  R  T   E  L  E   I  S  Y   A  P  R  L                            nsp4      5833       5843       5853       5863       5873       5883GACCAAGAAA AAGAAGAAUU ACUACGCAAG AAAUUACAGU UAAAUCCCAC ACCUGCUAAC  D  Q  E   K  E  E   L  L  R  K   K  L  Q   L  N  P   T  P  A  N                            nsp4      5893       5903       5913       5923       5933       5943AGAAGCAGAU ACCAGUCCAG GAAGGUGGAG AACAUGAAAG CCAUAACAGC UAGACGUAUU  R  S  R   Y  Q  S   R  K  V  E   N  M  K   A  I  T   A  R  R  I                            nsp4      5953       5963       5973       5983       5993       6003CUGCAAGGCC UAGGGCAUUA UUUGAAGGCA GAAGGAAAAG UGGAGUGCUA CCGAACCCUG  L  Q  G   L  G  H   Y  L  K  A   E  G  K   V  E  C   Y  R  T  L                            nsp4      6013       6023       6033       6043       6053       6063CAUCCUGUUC CUUUGUAUUC AUCUAGUGUG AACCGUGCCU UUUCAAGCCC CAAGGUCGCA  H  P  V   P  L  Y   S  S  S  V   N  R  A   F  S  S   P  K  V  A                            nsp4      6073       6083       6093       6103       6113       6123GUGGAAGCCU GUAACGCCAU GUUGAAAGAG AACUUUCCGA CUGUGGCUUC UUACUGUAUU  V  E  A   C  N  A   M  L  K  E   N  F  P   T  V  A   S  Y  C  I                            nsp4      6133       6143       6153       6163       6173       6183AUUCCAGAGU ACGAUGCCUA UUUGGACAUG GUUGACGGAG CUUCAUGCUG CUUAGACACU  I  P  E   Y  D  A   Y  L  D  M   V  D  G   A  S  C   C  L  D  T                            nsp4      6193       6203       6213       6223       6233       6243GCCAGUUUUU GCCCUGCAAA GCUGCGCAGC UUUCCAAAGA AACACUCCUA UUUGGAACCC  A  S  F   C  P  A   K  L  R  S   F  P  K   K  H  S   Y  L  E  P                            nsp4      6253       6263       6273       6283       6293       6303ACAAUACGAU CGGCAGUGCC UUCAGCGAUC CAGAACACGC UCCAGAACGU CCUGGCAGCU  T  I  R   S  A  V   P  S  A  I   Q  N  T   L  Q  N   V  L  A  A                            nsp4      6313       6323       6333       6343       6353       6363GCCACAAAAA GAAAUUGCAA UGUCACGCAA AUGAGAGAAU UGCCCGUAUU GGAUUCGGCG  A  T  K   R  N  C   N  V  T  Q   M  R  E   L  P  V   L  D  S  A                            nsp4      6373       6383       6393       6403       6413       6423GCCUUUAAUG UGGAAUGCUU CAAGAAAUAU GCGUGUAAUA AUGAAUAUUG GGAAACGUUU  A  F  N   V  E  C   F  K  K  Y   A  C  N   N  E  Y   W  E  T  F                            nsp4      6433       6443       6453       6463       6473       6483AAAGAAAACC CCAUCAGGCU UACUGAAGAA AACGUGGUAA AUUACAUUAC CAAAUUAAAA  K  E  N   P  I  R   L  T  E  E   N  V  V   N  Y  I   T  K  L  K                            nsp4      6493       6503       6513       6523       6533       6543GGACCAAAAG CUGCUGCUCU UUUUGCGAAG ACACAUAAUU UGAAUAUGUU GCAGGACAUA  G  P  K   A  A  A   L  F  A  K   T  H  N   L  N  M   L  Q  D  I                            nsp4      6553       6563       6573       6583       6593       6603CCAAUGGACA GGUUUGUAAU GGACUUAAAG AGAGACGUGA AAGUGACUCC AGGAACAAAA  P  M  D   R  F  V   M  D  L  K   R  D  V   K  V  T   P  G  T  K                            nsp4      6613       6623       6633       6643       6653       6663CAUACUGAAG AACGGCCCAA GGUACAGGUG AUCCAGGCUG CCGAUCCGCU AGCAACAGCG  H  T  E   E  R  P   K  V  Q  V   I  Q  A   A  D  P   L  A  T  A                            nsp4      6673       6683       6693       6703       6713       6723UAUCUGUGCG GAAUCCACCG AGAGCUGGUU AGGAGAUUAA AUGCGGUCCU GCUUCCGAAC  Y  L  C   G  I  H   R  E  L  V   R  R  L   N  A  V   L  L  P  N                            nsp4      6733       6743       6753       6763       6773       6783AUUCAUACAC UGUUUGAUAU GUCGGCUGAA GACUUUGACG CUAUUAUAGC CGAGCACUUC  I  H  T   L  E  D   M  S  A  E   D  F  D   A  I  I   A  E  H  F                            nsp4      6793       6803       6813       6823       6833       6843CAGCCUGGGG AUUGUGUUCU GGAAACUGAC AUCGCGUCGU UUGAUAAAAG UGAGGACGAC  Q  P  G   D  C  V   L  E  T  D   I  A  S   F  D  K   S  E  D  D                            nsp4      6853       6863       6873       6883       6893       6903GCCAUGGCUC UGACCGCGUU AAUGAUUCUG GAAGACUUAG GUGUGGACGC AGAGCUGUUG  A  M  A   L  T  A   L  M  I  L   E  D  L   G  V  D   A  E  L  L                            nsp4      6913       6923       6933       6943       6953       6963ACGCUGAUUG AGGCGGCUUU CGGCGAAAUU UCAUCAAUAC AUUUGCCCAC UAAAACUAAA  T  L  I   E  A  A   F  G  E  I   S  S  I   H  L  P   T  K  T  K                            nsp4      6973       6983       6993       7003       7013       7023UUUAAAUUCG GAGCCAUGAU GAAAUCUGGA AUGUUCCUCA CACUGUUUGU GAACACAGUC  F  K  F   G  A  M   M  K  S  G   M  F  L   T  L  F   V  N  T  V                            nsp4      7033       7043       7053       7063       7073       7083AUUAACAUUG UAAUCGCAAG CAGAGUGUUG AGAGAACGGC UAACCGGAUC ACCAUGUGCA  I  N  I   V  I  A   S  R  V  L   R  E  R   L  T  G   S  P  C  A                            nsp4      7093       7103       7113       7123       7133       7143GCAUUCAUUG GAGAUGACAA UAUCGUGAAA GGAGUCAAAU CGGACAAAUU AAUGGCAGAC  A  F  I   G  D  D   N  I  V  K   G  V  K   S  D  K   L  M  A  D                            nsp4      7153       7163       7173       7183       7193       7203AGGUGCGCCA CCUGGUUGAA UAUGGAAGUC AAGAUUAUAG AUGCUGUGGU GGGCGAGAAA  R  C  A   T  W  L   N  M  E  V   K  I  I   D  A  V   V  G  E  K                            nsp4      7213       7223       7233       7243       7253       7263GCGCCUUAUU UCUGUGGAGG GUUUAUUUUG UGUGACUCCG UGACCGGCAC AGCGUGCCGU  A  P  Y   F  C  G   G  E  I  L   C  D  S   V  T  G   T  A  C  R                            nsp4      7273       7283       7293       7303       7313       7323GUGGCAGACC CCCUAAAAAG GCUGUUUAAG CUAGGCAAAC CUCUGGCAGC AGACGAUGAA  V  A  D   P  L  K   R  L  F  K   L  G  K   P  L  A   A  D  D  E                            nsp4      7333       7343       7353       7363       7373       7383CAUGAUGAUG ACAGGAGAAG GGCAUUGCAU GAGGAGUCAA CACGCUGGAA CCGAGUGGGU  H  D  D   D  R  R   R  A  L  H   E  E  S   T  R  W   N  R  V  G                            nsp4      7393       7403 7      413       7423       7433       7443AUUCUUUCAG AGCUGUGCAA GGCAGUAGAA UCAAGGUAUG AAACCGUAGG AACUUCCAUC  I  L  S   E  L  C   K  A  V  E   S  R  Y   E  T  V   G  T  S  I                            nsp4      7453       7463       7473       7483       7493       7503AUAGUUAUGG CCAUGACUAC UCUAGCUAGC AGUGUUAAAU CAUUCAGCUA CCUGAGAGGG  I  V  M   A  M  T   T  L  A  S   S  V  K   S  F  S   Y  L  R  G                            nsp4       7513       7523 7527GCCCCUAUAA CUCUCUACGG CUAA   A  P  I   T  L  Y   G  *                            nsp4      7537       7547       7557       7567 7568CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAGACUAG U                          virUTR      7578       7588       7598       7608       7618       7628AUGUUUGUGU UUCUUGUGCU GCUGCCUCUU GUGUCUUCUC AGUGUGUGGU GAGAUUUCCA  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   V  R  F  P                          RBD (S protein)      7638       7648       7658       7668       7678       7688AAUAUUACAA AUCUGUGUCC AUUUGGAGAA GUGUUUAAUG CAACAAGAUU UGCAUCUGUG  N  I  T   N  L  C   P  F  G  E   V  F  N   A  T  R   F  A  S  V                          RBD (S protein)      7698       7708       7718       7728       7738       7748UAUGCAUGGA AUAGAAAAAG AAUUUCUAAU UGUGUGGCUG AUUAUUCUGU GCUGUAUAAU  Y  A  W   N  R  K   R  I  S  N   C  V  A   D  Y  S   V  L  Y  N                          RBD (S protein)      7758       7768       7778       7788       7798       7808AGUGCUUCUU UUUCCACAUU UAAAUGUUAU GGAGUGUCUC CAACAAAAUU AAAUGAUUUA  S  A  S   F  S  T   F  K  C  Y   G  V  S   P  T  K   L  N  D  L                          RBD (S protein)      7818       7828       7838       7848       7858       7868UGUUUUACAA AUGUGUAUGC UGAUUCUUUU GUGAUCAGAG GUGAUGAAGU GAGACAGAUU  C  F  T   N  V  Y   A  D  S  F   V  I  R   G  D  E   V  R  Q  I                          RBD (S protein)      7878       7888       7898       7908       7918       7928GCCCCCGGAC AGACAGGAAA AAUUGCUGAU UACAAUUACA AACUGCCUGA UGAUUUUACA  A  P  G   Q  T  G   K  I  A  D   Y  N  Y   K  L  P   D  D  F  T                          RBD (S protein)      7938       7948       7958       7968       7978       7988GGAUGUGUGA UUGCUUGGAA UUCUAAUAAU UUAGAUUCUA AAGUGGGAGG AAAUUACAAU  G  C  V   I  A  W   N  S  N  N   L  D  S   K  V  G   G  N  Y  N                          RBD (S protein)      7998       8008       8018       8028       8038       8048UAUCUGUACA GACUGUUUAG AAAAUCAAAU CUGAAACCUU UUGAAAGAGA UAUUUCAACA  Y  L  Y   R  L  F   R  K  S  N   L  K  P   F  E  R   D  I  S  T                          RBD (S protein)      8058       8068       8078       8088       8098       8108GAAAUUUAUC AGGCUGGAUC AACACCUUGU AAUGGAGUGG AAGGAUUUAA UUGUUAUUUU  E  I  Y   Q  A  G   S  T  P  C   N  G  V   E  G  F   N  C  Y  F                          RBD (S protein)      8118       8128       8138       8148       8158       8168CCAUUACAGA GCUAUGGAUU UCAGCCAACC AAUGGUGUGG GAUAUCAGCC AUAUAGAGUG  P  L  Q   S  Y  G   E  Q  P  T   N  G  V   G  Y  Q   P  Y  R  V                          RBD (S protein)      8178       8188       8198       8208       8218   8222GUGGUGCUGU CUUUUGAACU GCUGCAUGCA CCUGCAACAG UGUGUGGACC UAAA  V  V  L   S  F  E   L  L  H  A   P  A  T   V  C  G   P  K                          RBD (S protein)      8232       8242       8249 GGCUCCCCCG GCUCCGGCUC CGGAUCU  G  S  P   G  S  G   S  G  S                           GS linker      8259       8269       8279       8289       8299       8309GGUUAUAUUC CUGAAGCUCC AAGAGAUGGG CAAGCUUACG UUCGUAAAGA UGGCGAAUGG  G  Y  I   P  E  A   P  R  D  G   Q  A  Y   V  R  K   D  G  E  W                          fibritin       8319       8329 8330GUAUUACUUU CUACCUUUUU A   V  L  L   S  T  E   L                          fibritin       8340   8345 GGAAGCGGCA GCGGA  G  S  G   S  G GS linker      8355       8365       8375       8385       8395       8405UCUGAACAGU ACAUUAAAUG GCCUUGGUAC AUUUGGCUUG GAUUUAUUGC AGGAUUAAUU  S  E  Q   Y  I  K   W  P  W  Y   I  W  L   G  F  I   A  G  L  I                            TM      8415       8425       8435       8445       8455       8465GCAAUUGUGA UGGUGACAAU UAUGUUAUGU UGUAUGACAU CAUGUUGUUC UUGUUUAAAA  A  I  V   M  V  T   I  M  L  C   C  M  T   S  C  C   S  C  L  K                            TM      8475       8485       8495       8505       8515       8525GGAUGUUGUU CUUGUGGAAG CUGUUGUAAA UUUGAUGAAG AUGAUUCUGA ACCUGUGUUA  G  C  C   S  C  G   S  C  C  K   F  D  E   D  D  S   E  P  V  L                            TM       8535       8545       8555AAAGGAGUGA AAUUGCAUUA CACAUGAUGA   K  G  V   K  L  H   Y  T  *  *                            TM      8565       8575       8585       8595       8605       8615CUCGAGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                                 FI element      8625       8635       8645       8655       8665       8675AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                                 FI element      8685       8695       8705       8715       8725       8735UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                                 FI element      8745       8755       8765       8775       8785       8795CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                                 FI element      8805       8815       8825       8835       8845       8855GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCGCGGCC GCAUGAAUAC                                 FI element      8865       8875       8885       8895       8905       8915AGCAGCAAUU GGCAAGCUGC UUACAUAGAA CUCGCGGCGA UUGGCAUGCC GCCUUAAAAU                                 FI element      8925       8935       8945       8955       8965   8969UUUUAUUUUA UUUUUUCUUU UCUUUUCCGA AUCGGAUUUU GUUUUUAAUA UUUC                                 FI element      8979       8989       8999       9009       9019       9029AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                                 Poly(A)      9039       9049       9059       9069       9079AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                                 Poly(A)

In some embodiments, vaccine RNA described herein comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 15, 16, 17,19, 20, 21, 24, 25, 26, 27, 30, and 32. A particularly preferred vaccineRNA described herein comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 15, 17, 19, 21, 25, 26, 30, and 32 suchas selected from the group consisting of SEQ ID NO: 17, 19, 21, 26, 30,and 32.

RNA described herein is preferably formulated in lipid nanoparticles(LNP). In one embodiment, the LNP comprise a cationic lipid, a neutrallipid, a steroid, a polymer conjugated lipid; and the RNA. In oneembodiment, the cationic lipid is ALC-0315, the neutral lipid is DSPC,the steroid is cholesterol, and the polymer conjugated lipid isALC-0159. The preferred mode of administration is intramuscularadministration, more preferably in aqueous cryoprotectant buffer forintramuscular administration. The drug product is a preferably apreservative-free, sterile dispersion of RNA formulated in lipidnanoparticles (LNP) in aqueous cryoprotectant buffer for intramuscularadministration.

In different embodiments, the drug product comprises the componentsshown below, preferably at the proportions or concentrations shownbelow:

Component Function Proportion (mol %) ALC-0315 _([1]) Functional lipid47.5 ALC-O159 _([2]) Functional lipid 1.8 DSPC _([3]) Structural lipid10.0 Cholesterol, synthetic Structural lipid 40.7 Component FunctionConcentration (mg/mL) Drug Substance Active 0.5 ALC-0315 _([1])Functional lipid 7.17 ALC-0159 _([2]) Functional lipid 0.89 DSPC _([3])Structural lipid 1.56 Cholesterol, synthetic Structural lipid 3.1Sucrose Cryoprotectant 102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na₂HPO₄Buffer 1.08 KH₂PO₄ Buffer 0.18 Water for injection Solvent/Vehicle q.s.Drug Substance Active 1.0 ALC-0315 _([1]) Functional lipid 13.56ALC-0159 _([2]) Functional lipid 1.77 DSPC _([3]) Structural lipid 3.11Cholesterol, synthetic Structural lipid 6.20 Sucrose Cryoprotectant102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na₂HPO₄ Buffer 1.08 KH₂PO₄ Buffer0.15 Water for injection Solvent/Vehicle q.s. _([1]) ALC-0315 =((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)/6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl2-hexyldecanoate _([2]) ALC-0159 = 2-[(polyethyleneglycol)-2000]-N,N-ditetradecylacetamide/2-[2-(ω-methoxy(polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide _([3]) DSPC =1,2-Distearoyl-sn-glycero-3-phosphocholine q.s. = quantum satis (as muchas may suffice)

In one embodiment, the ratio of mRNA to total lipid (N/P) is between 6.0and 6.5 such as about 6.0 or about 6.3.

Nucleic Acid Containing Particles

Nucleic acids described herein such as RNA encoding a vaccine antigenmay be administered formulated as particles.

In the context of the present disclosure, the term “particle” relates toa structured entity formed by molecules or molecule complexes. In oneembodiment, the term “particle” relates to a micro- or nano-sizedstructure, such as a micro- or nano-sized compact structure dispersed ina medium. In one embodiment, a particle is a nucleic acid containingparticle such as a particle comprising DNA, RNA or a mixture thereof.

Electrostatic interactions between positively charged molecules such aspolymers and lipids and negatively charged nucleic acid are involved inparticle formation. This results in complexation and spontaneousformation of nucleic acid particles. In one embodiment, a nucleic acidparticle is a nanoparticle.

As used in the present disclosure, “nanoparticle” refers to a particlehaving an average diameter suitable for parenteral administration.

A “nucleic acid particle” can be used to deliver nucleic acid to atarget site of interest (e.g., cell, tissue, organ, and the like). Anucleic acid particle may be formed from at least one cationic orcationically ionizable lipid or lipid-like material, at least onecationic polymer such as protamine, or a mixture thereof and nucleicacid. Nucleic acid particles include lipid nanoparticle (LNP)-based andlipoplex (LPX)-based formulations.

Without intending to be bound by any theory, it is believed that thecationic or cationically ionizable lipid or lipid-like material and/orthe cationic polymer combine together with the nucleic acid to formaggregates, and this aggregation results in colloidally stableparticles.

In one embodiment, particles described herein further comprise at leastone lipid or lipid-like material other than a cationic or cationicallyionizable lipid or lipid-like material, at least one polymer other thana cationic polymer, or a mixture thereof

In some embodiments, nucleic acid particles comprise more than one typeof nucleic acid molecules, where the molecular parameters of the nucleicacid molecules may be similar or different from each other, like withrespect to molar mass or fundamental structural elements such asmolecular architecture, capping, coding regions or other features,Nucleic acid particles described herein may have an average diameterthat in one embodiment ranges from about 30 nm to about 1000 nm, fromabout 50 nm to about 800 nm, from about 70 nm to about 600 nm, fromabout 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

Nucleic acid particles described herein may exhibit a polydispersityindex less than about 0.5, less than about 0.4, less than about 0.3, orabout 0.2 or less. By way of example, the nucleic acid particles canexhibit a polydispersity index in a range of about 0.1 to about 0.3 orabout 0.2 to about 0.3.

With respect to RNA lipid particles, the N/P ratio gives the ratio ofthe nitrogen groups in the lipid to the number of phosphate groups inthe RNA. It is correlated to the charge ratio, as the nitrogen atoms(depending on the pH) are usually positively charged and the phosphategroups are negatively charged. The N/P ratio, where a charge equilibriumexists, depends on the pH. Lipid formulations are frequently formed atN/P ratios larger than four up to twelve, because positively chargednanoparticles are considered favorable for transfection. In that case,RNA is considered to be completely bound to nanoparticles.

Nucleic acid particles described herein can be prepared using a widerange of methods that may involve obtaining a colloid from at least onecationic or cationically ionizable lipid or lipid-like material and/orat least one cationic polymer and mixing the colloid with nucleic acidto obtain nucleic acid particles.

The term “colloid” as used herein relates to a type of homogeneousmixture in which dispersed particles do not settle out. The insolubleparticles in the mixture are microscopic, with particle sizes between 1and 1000 nanometers. The mixture may be termed a colloid or a colloidalsuspension. Sometimes the term “colloid” only refers to the particles inthe mixture and not the entire suspension.

For the preparation of colloids comprising at least one cationic orcationically ionizable lipid or lipid-like material and/or at least onecationic polymer methods are applicable herein that are conventionallyused for preparing liposomal vesicles and are appropriately adapted. Themost commonly used methods for preparing liposomal vesicles share thefollowing fundamental stages: (i) lipids dissolution in organicsolvents, (ii) drying of the resultant solution, and (iii) hydration ofdried lipid (using various aqueous media).

In the film hydration method, lipids are firstly dissolved in a suitableorganic solvent, and dried down to yield a thin film at the bottom ofthe flask. The obtained lipid film is hydrated using an appropriateaqueous medium to produce a liposomal dispersion. Furthermore, anadditional downsizing step may be included.

Reverse phase evaporation is an alternative method to the film hydrationfor preparing liposomal vesicles that involves formation of awater-in-oil emulsion between an aqueous phase and an organic phasecontaining lipids. A brief sonication of this mixture is required forsystem homogenization. The removal of the organic phase under reducedpressure yields a milky gel that turns subsequently into a liposomalsuspension.

The term “ethanol injection technique” refers to a process, in which anethanol solution comprising lipids is rapidly injected into an aqueoussolution through a needle. This action disperses the lipids throughoutthe solution and promotes lipid structure formation, for example lipidvesicle formation such as liposome formation. Generally, the RNAlipoplex particles described herein are obtainable by adding RNA to acolloidal liposome dispersion. Using the ethanol injection technique,such colloidal liposome dispersion is, in one embodiment, formed asfollows: an ethanol solution comprising lipids, such as cationic lipidsand additional lipids, is injected into an aqueous solution understirring. In one embodiment, the RNA lipoplex particles described hereinare obtainable without a step of extrusion.

The term “extruding” or “extrusion” refers to the creation of particleshaving a fixed, cross-sectional profile. In particular, it refers to thedownsizing of a particle, whereby the particle is forced through filterswith defined pores.

Other methods having organic solvent free characteristics may also beused according to the present disclosure for preparing a colloid.

LNPs typically comprise four components: ionizable cationic lipids,neutral lipids such as phospholipids, a steroid such as cholesterol, anda polymer conjugated lipid such as polyethylene glycol (PEG)-lipids.Each component is responsible for payload protection, and enableseffective intracellular delivery. LNPs may be prepared by mixing lipidsdissolved in ethanol rapidly with nucleic acid in an aqueous buffer.

The term “average diameter” refers to the mean hydrodynamic diameter ofparticles as measured by dynamic laser light scattering (DLS) with dataanalysis using the so-called cumulant algorithm, which provides asresults the so-called Z_(average) with the dimension of a length, andthe polydispersity index (PI), which is dimensionless (Koppel, D., J.Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter”,“diameter” or “size” for particles is used synonymously with this valueof the Z_(average).

The “polydispersity index” is preferably calculated based on dynamiclight scattering measurements by the so-called cumulant analysis asmentioned in the definition of the “average diameter”. Under certainprerequisites, it can be taken as a measure of the size distribution ofan ensemble of nanoparticles.

Different types of nucleic acid containing particles have been describedpreviously to be suitable for delivery of nucleic acid in particulateform (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). Fornon-viral nucleic acid delivery vehicles, nanoparticle encapsulation ofnucleic acid physically protects nucleic acid from degradation and,depending on the specific chemistry, can aid in cellular uptake andendosomal escape.

The present disclosure describes particles comprising nucleic acid, atleast one cationic or cationically ionizable lipid or lipid-likematerial, and/or at least one cationic polymer which associate withnucleic acid to form nucleic acid particles and compositions comprisingsuch particles. The nucleic acid particles may comprise nucleic acidwhich is complexed in different forms by non-covalent interactions tothe particle. The particles described herein are not viral particles, inparticular infectious viral particles, i.e., they are not able tovirally infect cells. Suitable cationic or cationically ionizable lipidsor lipid-like materials and cationic polymers are those that formnucleic acid particles and are included by the term “particle formingcomponents” or “particle forming agents”. The term “particle formingcomponents” or “particle forming agents” relates to any components whichassociate with nucleic acid to form nucleic acid particles. Suchcomponents include any component which can be part of nucleic acidparticles.

Cationic Polymer

Given their high degree of chemical flexibility, polymers are commonlyused materials for nanoparticle-based delivery. Typically, cationicpolymers are used to electrostatically condense the negatively chargednucleic acid into nanoparticles. These positively charged groups oftenconsist of amines that change their state of protonation in the pH rangebetween 5.5 and 7.5, thought to lead to an ion imbalance that results inendosomal rupture. Polymers such as poly-L-lysine, polyamidoamine,protamine and polyethyleneimine, as well as naturally occurring polymerssuch as chitosan have all been applied to nucleic acid delivery and aresuitable as cationic polymers herein. In addition, some investigatorshave synthesized polymers specifically for nucleic acid delivery.Poly(S3-amino esters), in particular, have gained widespread use innucleic acid delivery owing to their ease of synthesis andbiodegradability. Such synthetic polymers are also suitable as cationicpolymers herein.

A “polymer,” as used herein, is given its ordinary meaning, i.e., amolecular structure comprising one or more repeat units (monomers),connected by covalent bonds. The repeat units can all be identical, orin some cases, there can be more than one type of repeat unit presentwithin the polymer. In some cases, the polymer is biologically derived,i.e., a biopolymer such as a protein. In some cases, additional moietiescan also be present in the polymer, for example targeting moieties suchas those described herein.

If more than one type of repeat unit is present within the polymer, thenthe polymer is said to be a “copolymer.” It is to be understood that thepolymer being employed herein can be a copolymer. The repeat unitsforming the copolymer can be arranged in any fashion. For example, therepeat units can be arranged in a random order, in an alternating order,or as a “block” copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers can have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

In certain embodiments, the polymer is biocompatible. Biocompatiblepolymers are polymers that typically do not result in significant celldeath at moderate concentrations. In certain embodiments, thebiocompatible polymer is biodegradable, i.e., the polymer is able todegrade, chemically and/or biologically, within a physiologicalenvironment, such as within the body.

In certain embodiments, polymer may be protamine or polyalkyleneimine,in particular protamine.

The term “protamine” refers to any of various strongly basic proteins ofrelatively low molecular weight that are rich in arginine and are foundassociated especially with DNA in place of somatic histones in the spermcells of various animals (as fish). In particular, the term “protamine”refers to proteins found in fish sperm that are strongly basic, aresoluble in water, are not coagulated by heat, and yield chiefly arginineupon hydrolysis. In purified form, they are used in a long-actingformulation of insulin and to neutralize the anticoagulant effects ofheparin.

According to the disclosure, the term “protamine” as used herein ismeant to comprise any protamine amino acid sequence obtained or derivedfrom natural or biological sources including fragments thereof andmultimeric forms of said amino acid sequence or fragment thereof as wellas (synthesized) polypeptides which are artificial and specificallydesigned for specific purposes and cannot be isolated from native orbiological sources.

In one embodiment, the polyalkyleneimine comprises polyethylenimineand/or polypropylenimine, preferably polyethyleneimine. A preferredpolyalkyleneimine is polyethyleneimine (PEI). The average molecularweight of PEI is preferably 0.75·10² to 10⁷ Da, preferably 1000 to 10⁵Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000Da, even more preferably 20000 to 25000 Da.

Preferred according to the disclosure is linear polyalkyleneimine suchas linear polyethyleneimine (PEI).

Cationic polymers (including polycationic polymers) contemplated for useherein include any cationic polymers which are able to electrostaticallybind nucleic acid. In one embodiment, cationic polymers contemplated foruse herein include any cationic polymers with which nucleic acid can beassociated, e.g. by forming complexes with the nucleic acid or formingvesicles in which the nucleic acid is enclosed or encapsulated.

Particles described herein may also comprise polymers other thancationic polymers, i.e., non-cationic polymers and/or anionic polymers.Collectively, anionic and neutral polymers are referred to herein asnon-cationic polymers.

Lipid and Lipid-Like Material

The terms “lipid” and “lipid-like material” are broadly defined hereinas molecules which comprise one or more hydrophobic moieties or groupsand optionally also one or more hydrophilic moieties or groups.Molecules comprising hydrophobic moieties and hydrophilic moieties arealso frequently denoted as amphiphiles. Lipids are usually poorlysoluble in water. In an aqueous environment, the amphiphilic natureallows the molecules to self-assemble into organized structures anddifferent phases. One of those phases consists of lipid bilayers, asthey are present in vesicles, multilamellar/unilamellar liposomes, ormembranes in an aqueous environment. Hydrophobicity can be conferred bythe inclusion of apolar groups that include, but are not limited to,long-chain saturated and unsaturated aliphatic hydrocarbon groups andsuch groups substituted by one or more aromatic, cycloaliphatic, orheterocyclic group(s). The hydrophilic groups may comprise polar and/orcharged groups and include carbohydrates, phosphate, carboxylic,sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

As used herein, the term “amphiphilic” refers to a molecule having botha polar portion and a non-polar portion. Often, an amphiphilic compoundhas a polar head attached to a long hydrophobic tail. In someembodiments, the polar portion is soluble in water, while the non-polarportion is insoluble in water. In addition, the polar portion may haveeither a formal positive charge, or a formal negative charge.Alternatively, the polar portion may have both a formal positive and anegative charge, and be a zwitterion or inner salt. For purposes of thedisclosure, the amphiphilic compound can be, but is not limited to, oneor a plurality of natural or non-natural lipids and lipid-likecompounds.

The term “lipid-like material”, “lipid-like compound” or “lipid-likemolecule” relates to substances that structurally and/or functionallyrelate to lipids but may not be considered as lipids in a strict sense.For example, the term includes compounds that are able to formamphiphilic layers as they are present in vesicles,multilamellar/unilamellar liposomes, or membranes in an aqueousenvironment and includes surfactants, or synthesized compounds with bothhydrophilic and hydrophobic moieties. Generally speaking, the termrefers to molecules, which comprise hydrophilic and hydrophobic moietieswith different structural organization, which may or may not be similarto that of lipids. As used herein, the term “lipid” is to be construedto cover both lipids and lipid-like materials unless otherwise indicatedherein or clearly contradicted by context.

Specific examples of amphiphilic compounds that may be included in anamphiphilic layer include, but are not limited to, phospholipids,aminolipids and sphingolipids.

In certain embodiments, the amphiphilic compound is a lipid. The term“lipid” refers to a group of organic compounds that are characterized bybeing insoluble in water, but soluble in many organic solvents.Generally, lipids may be divided into eight categories: fatty acids,glycerolipids, glycerophospholipids, sphingolipids, saccharolipids,polyketides (derived from condensation of ketoacyl subunits), sterollipids and prenol lipids (derived from condensation of isoprenesubunits). Although the term “lipid” is sometimes used as a synonym forfats, fats are a subgroup of lipids called triglycerides. Lipids alsoencompass molecules such as fatty acids and their derivatives (includingtri-, di-, monoglycerides, and phospholipids), as well assterol-containing metabolites such as cholesterol.

Fatty acids, or fatty acid residues are a diverse group of moleculesmade of a hydrocarbon chain that terminates with a carboxylic acidgroup; this arrangement confers the molecule with a polar, hydrophilicend, and a nonpolar, hydrophobic end that is insoluble in water. Thecarbon chain, typically between four and 24 carbons long, may besaturated or unsaturated, and may be attached to functional groupscontaining oxygen, halogens, nitrogen, and sulfur. If a fatty acidcontains a double bond, there is the possibility of either a cis ortrans geometric isomerism, which significantly affects the molecule'sconfiguration. Cis-double bonds cause the fatty acid chain to bend, aneffect that is compounded with more double bonds in the chain. Othermajor lipid classes in the fatty acid category are the fatty esters andfatty amides. Glycerolipids are composed of mono-, di-, andtri-substituted glycerols, the best-known being the fatty acid triestersof glycerol, called triglycerides. The word “triacylglycerol” issometimes used synonymously with “triglyceride”. In these compounds, thethree hydroxyl groups of glycerol are each esterified, typically bydifferent fatty acids. Additional subclasses of glycerolipids arerepresented by glycosylglycerols, which are characterized by thepresence of one or more sugar residues attached to glycerol via aglycosidic linkage.

The glycerophospholipids are amphipathic molecules (containing bothhydrophobic and hydrophilic regions) that contain a glycerol core linkedto two fatty acid-derived “tails” by ester linkages and to one “head”group by a phosphate ester linkage. Examples of glycerophospholipids,usually referred to as phospholipids (though sphingomyelins are alsoclassified as phospholipids) are phosphatidylcholine (also known as PC,GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) andphosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a commonstructural feature, a sphingoid base backbone. The major sphingoid basein mammals is commonly referred to as sphingosine. Ceramides(N-acyl-sphingoid bases) are a major subclass of sphingoid basederivatives with an amide-linked fatty acid. The fatty acids aretypically saturated or mono-unsaturated with chain lengths from 16 to 26carbon atoms. The major phosphosphingolipids of mammals aresphingomyelins (ceramide phosphocholines), whereas insects containmainly ceramide phosphoethanolamines and fungi have phytoceramidephosphoinositols and mannose-containing headgroups. Theglycosphingolipids are a diverse family of molecules composed of one ormore sugar residues linked via a glycosidic bond to the sphingoid base.Examples of these are the simple and complex glycosphingolipids such ascerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives, or tocopheroland its derivatives, are an important component of membrane lipids,along with the glycerophospholipids and sphingomyelins.

Saccharolipids describe compounds in which fatty acids are linkeddirectly to a sugar backbone, forming structures that are compatiblewith membrane bilayers. In the saccharolipids, a monosaccharidesubstitutes for the glycerol backbone present in glycerolipids andglycerophospholipids. The most familiar saccharolipids are the acylatedglucosamine precursors of the Lipid A component of thelipopolysaccharides in Gram-negative bacteria. Typical lipid A moleculesare disaccharides of glucosamine, which are derivatized with as many asseven fatty-acyl chains. The minimal lipopolysaccharide required forgrowth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide ofglucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonicacid (Kdo) residues.

Polyketides are synthesized by polymerization of acetyl and propionylsubunits by classic enzymes as well as iterative and multimodularenzymes that share mechanistic features with the fatty acid synthases.They comprise a large number of secondary metabolites and naturalproducts from animal, plant, bacterial, fungal and marine sources, andhave great structural diversity. Many polyketides are cyclic moleculeswhose backbones are often further modified by glycosylation,methylation, hydroxylation, oxidation, or other processes.

According to the disclosure, lipids and lipid-like materials may becationic, anionic or neutral. Neutral lipids or lipid-like materialsexist in an uncharged or neutral zwitterionic form at a selected pH.

Cationic or Cationically Ionizable Lipids or Lipid-Like Materials

The nucleic acid particles described herein may comprise at least onecationic or cationically ionizable lipid or lipid-like material asparticle forming agent. Cationic or cationically ionizable lipids orlipid-like materials contemplated for use herein include any cationic orcationically ionizable lipids or lipid-like materials which are able toelectrostatically bind nucleic acid. In one embodiment, cationic orcationically ionizable lipids or lipid-like materials contemplated foruse herein can be associated with nucleic acid, e.g. by formingcomplexes with the nucleic acid or forming vesicles in which the nucleicacid is enclosed or encapsulated.

As used herein, a “cationic lipid” or “cationic lipid-like material”refers to a lipid or lipid-like material having a net positive charge.Cationic lipids or lipid-like materials bind negatively charged nucleicacid by electrostatic interaction. Generally, cationic lipids possess alipophilic moiety, such as a sterol, an acyl chain, a diacyl or moreacyl chains, and the head group of the lipid typically carries thepositive charge.

In certain embodiments, a cationic lipid or lipid-like material has anet positive charge only at certain pH, in particular acidic pH, whileit has preferably no net positive charge, preferably has no charge,i.e., it is neutral, at a different, preferably higher pH such asphysiological pH. This ionizable behavior is thought to enhance efficacythrough helping with endosomal escape and reducing toxicity as comparedwith particles that remain cationic at physiological pH. For purposes ofthe present disclosure, such “cationically ionizable” lipids orlipid-like materials are comprised by the term “cationic lipid orlipid-like material” unless contradicted by the circumstances.

In one embodiment, the cationic or cationically ionizable lipid orlipid-like material comprises a head group which includes at least onenitrogen atom (N) which is positive charged or capable of beingprotonated.

Examples of cationic lipids include, but are not limited to1,2-dioleoyl-3-trimethylammonium propane (DOTAP);N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),dimethyldioctadecylammonium (DDAB);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);1,2-diacyloxy-3-dimethylammonium propanes;1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammoniumchloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),I,2-dimyristoyl-3-trimethylammonium propane (DMTAP),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-I-propanamium trifluoroacetate (DOSPA),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate(DLin-MC3-DMA),N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (DMRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminiumbromide (GAP-DMORIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (GAP-DLRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (GAP-DMRIE),N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (βAE-DMRIE),N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(DOBAQ),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP),1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amoniumbromide (DLRIE),N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminiumbromide (DMORIE), di((Z)-non-2-en-1-yl)8,8′-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate(ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA),N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA),Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate(L319),N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide(lipidoid 98N₁₂-5),1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol(lipidoid C12-200).

In some embodiments, the cationic lipid may comprise from about 10 mol %to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % toabout 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % toabout 100 mol % of the total lipid present in the particle.

Additional Lipids or Lipid-Like Materials

Particles described herein may also comprise lipids or lipid-likematerials other than cationic or cationically ionizable lipids orlipid-like materials, i.e., non-cationic lipids or lipid-like materials(including non-cationically ionizable lipids or lipid-like materials).Collectively, anionic and neutral lipids or lipid-like materials arereferred to herein as non-cationic lipids or lipid-like materials.Optimizing the formulation of nucleic acid particles by addition ofother hydrophobic moieties, such as cholesterol and lipids, in additionto an ionizable/cationic lipid or lipid-like material may enhanceparticle stability and efficacy of nucleic acid delivery.

An additional lipid or lipid-like material may be incorporated which mayor may not affect the overall charge of the nucleic acid particles. Incertain embodiments, the additional lipid or lipid-like material is anon-cationic lipid or lipid-like material. The non-cationic lipid maycomprise, e.g., one or more anionic lipids and/or neutral lipids. Asused herein, an “anionic lipid” refers to any lipid that is negativelycharged at a selected pH. As used herein, a “neutral lipid” refers toany of a number of lipid species that exist either in an uncharged orneutral zwitterionic form at a selected pH. In preferred embodiments,the additional lipid comprises one of the following neutral lipidcomponents: (1) a phospholipid, (2) cholesterol or a derivative thereof;or (3) a mixture of a phospholipid and cholesterol or a derivativethereof. Examples of cholesterol derivatives include, but are notlimited to, cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,tocopherol and derivatives thereof, and mixtures thereof.

Specific phospholipids that can be used include, but are not limited to,phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols,phosphatidic acids, phosphatidylserines or sphingomyelin. Suchphospholipids include in particular diacylphosphatidylcholines, such asdistearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dimyristoylphosphatidylcholine (DMPC),dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine(DAPC), dibehenoylphosphatidylcholine (DBPC),ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine(DLPC), palmitoyloleoyl-phosphatidylcholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) andphosphatidylethanolamines, in particulardiacylphosphatidylethanolamines, such asdioleoylphosphatidylethanolamine (DOPE),distearoyl-phosphatidylethanolamine (DSPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),dilauroyl-phosphatidylethanolamine (DLPE),diphytanoyl-phosphatidylethanolamine (DPyPE), and furtherphosphatidylethanolamine lipids with different hydrophobic chains.

In certain preferred embodiments, the additional lipid is DSPC or DSPCand cholesterol.

In certain embodiments, the nucleic acid particles include both acationic lipid and an additional lipid.

In one embodiment, particles described herein include a polymerconjugated lipid such as a pegylated lipid. The term “pegylated lipid”refers to a molecule comprising both a lipid portion and a polyethyleneglycol portion. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of the at least onecationic lipid compared to the amount of the at least one additionallipid may affect important nucleic acid particle characteristics, suchas charge, particle size, stability, tissue selectivity, and bioactivityof the nucleic acid. Accordingly, in some embodiments, the molar ratioof the at least one cationic lipid to the at least one additional lipidis from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 toabout 1:1.

In some embodiments, the non-cationic lipid, in particular neutrallipid, (e.g., one or more phospholipids and/or cholesterol) may comprisefrom about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol%, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60mol %, or from about 0 mol % to about 50 mol %, of the total lipidpresent in the particle.

Lipoplex Particles

In certain embodiments of the present disclosure, the RNA describedherein may be present in RNA lipoplex particles.

In the context of the present disclosure, the term “RNA lipoplexparticle” relates to a particle that contains lipid, in particularcationic lipid, and RNA. Electrostatic interactions between positivelycharged liposomes and negatively charged RNA results in complexation andspontaneous formation of RNA lipoplex particles. Positively chargedliposomes may be generally synthesized using a cationic lipid, such asDOTMA, and additional lipids, such as DOPE. In one embodiment, a RNAlipoplex particle is a nanoparticle.

In certain embodiments, the RNA lipoplex particles include both acationic lipid and an additional lipid. In an exemplary embodiment, thecationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipidto the at least one additional lipid is from about 10:0 to about 1:9,about 4:1 to about 1:2, or about 3:1 to about 1:1. In specificembodiments, the molar ratio may be about 3:1, about 2.75:1, about2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1,or about 1:1. In an exemplary embodiment, the molar ratio of the atleast one cationic lipid to the at least one additional lipid is about2:1.

RNA lipoplex particles described herein have an average diameter that inone embodiment ranges from about 200 nm to about 1000 nm, from about 200nm to about 800 nm, from about 250 to about 700 nm, from about 400 toabout 600 nm, from about 300 nm to about 500 nm, or from about 350 nm toabout 400 nm. In specific embodiments, the RNA lipoplex particles havean average diameter of about 200 nm, about 225 nm, about 250 nm, about275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment,the RNA lipoplex particles have an average diameter that ranges fromabout 250 nm to about 700 nm. In another embodiment, the RNA lipoplexparticles have an average diameter that ranges from about 300 nm toabout 500 nm. In an exemplary embodiment, the RNA lipoplex particleshave an average diameter of about 400 nm.

The RNA lipoplex particles and compositions comprising RNA lipoplexparticles described herein are useful for delivery of RNA to a targettissue after parenteral administration, in particular after intravenousadministration. The RNA lipoplex particles may be prepared usingliposomes that may be obtained by injecting a solution of the lipids inethanol into water or a suitable aqueous phase. In one embodiment, theaqueous phase has an acidic pH. In one embodiment, the aqueous phasecomprises acetic acid, e.g., in an amount of about 5 mM. Liposomes maybe used for preparing RNA lipoplex particles by mixing the liposomeswith RNA. In one embodiment, the liposomes and RNA lipoplex particlescomprise at least one cationic lipid and at least one additional lipid.In one embodiment, the at least one cationic lipid comprises1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, theat least one additional lipid comprises1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC). In one embodiment, the at least one cationic lipid comprises1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the atleast one additional lipid comprises1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In oneembodiment, the liposomes and RNA lipoplex particles comprise1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). Spleentargeting RNA lipoplex particles are described in WO 2013/143683, hereinincorporated by reference. It has been found that RNA lipoplex particleshaving a net negative charge may be used to preferentially target spleentissue or spleen cells such as antigen-presenting cells, in particulardendritic cells. Accordingly, following administration of the RNAlipoplex particles, RNA accumulation and/or RNA expression in the spleenoccurs. Thus, RNA lipoplex particles of the disclosure may be used forexpressing RNA in the spleen. In an embodiment, after administration ofthe RNA lipoplex particles, no or essentially no RNA accumulation and/orRNA expression in the lung and/or liver occurs. In one embodiment, afteradministration of the RNA lipoplex particles, RNA accumulation and/orRNA expression in antigen presenting cells, such as professional antigenpresenting cells in the spleen occurs. Thus, RNA lipoplex particles ofthe disclosure may be used for expressing RNA in such antigen presentingcells. In one embodiment, the antigen presenting cells are dendriticcells and/or macrophages.

Lipid Nanoparticles (LNPs)

In one embodiment, nucleic acid such as RNA described herein isadministered in the form of lipid nanoparticles (LNPs). The LNP maycomprise any lipid capable of forming a particle to which the one ormore nucleic acid molecules are attached, or in which the one or morenucleic acid molecules are encapsulated.

In one embodiment, the LNP comprises one or more cationic lipids, andone or more stabilizing lipids. Stabilizing lipids include neutrallipids and pegylated lipids.

In one embodiment, the LNP comprises a cationic lipid, a neutral lipid,a steroid, a polymer conjugated lipid; and the RNA, encapsulated withinor associated with the lipid nanoparticle. In one embodiment, the LNPcomprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent,from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 molpercent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, theLNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7,47.8, 47.9 or 48.0 mol percent of the cationic lipid.

In one embodiment, the neutral lipid is present in a concentrationranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to11 mol percent. In one embodiment, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent.

In one embodiment, the steroid is present in a concentration rangingfrom 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43mol percent. In one embodiment, the steroid is present in aconcentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.

In one embodiment, the LNP comprises from 1 to 10 mol percent, from 1 to5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugatedlipid.

In one embodiment, the LNP comprises from 40 to 50 mol percent acationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to45 mol percent of a steroid; from 1 to 10 mol percent of a polymerconjugated lipid; and the RNA, encapsulated within or associated withthe lipid nanoparticle.

In one embodiment, the mol percent is determined based on total mol oflipid present in the lipid nanoparticle.

In one embodiment, the neutral lipid is selected from the groupconsisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE,DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid isselected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPEand SM. In one embodiment, the neutral lipid is DSPC.

In one embodiment, the steroid is cholesterol.

In one embodiment, the polymer conjugated lipid is a pegylated lipid. Inone embodiment, the pegylated lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:R¹² and R¹³ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and w has a mean value ranging from 30 to 60. In one embodiment, R¹² andR¹³ are each independently straight, saturated alkyl chains containingfrom 12 to 16 carbon atoms. In one embodiment, w has a mean valueranging from 40 to 55. In one embodiment, the average w is about 45. Inone embodiment, R¹² and R¹³ are each independently a straight, saturatedalkyl chain containing about 14 carbon atoms, and w has a mean value ofabout 45.

In one embodiment, the pegylated lipid is DMG-PEG 2000, e.g., having thefollowing structure:

In some embodiments, the cationic lipid component of the LNPs has thestructure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:one of L¹ or L² is —O(C═O)—, —(C═O)—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—,—(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond;G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂alkenylene;G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈cycloalkenylene;R^(a) is H or C₁-C₁₂ alkyl;R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;R⁴ is C₁-C₁₂ alkyl;R⁵ is H or C₁-C₆ alkyl; andx is 0, 1 or 2.

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following structures (IIIA) or (IIIB):

wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of Formula (III), the lipid hasstructure (IIIA), and in other embodiments, the lipid has structure(IIIB).

In other embodiments of Formula (III), the lipid has one of thefollowing structures (IIIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments of Formula (III), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—. In some different embodiments of any of the foregoing, L andL² are each independently —(C═O)O— or —O(C═O)—. For example, in someembodiments each of L¹ and L² is —(C═O)O—.

In some different embodiments of Formula (III), the lipid has one of thefollowing structures (IIIE) or (IIIF):

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integerranging from 2 to 12, for example from 2 to 8 or from 2 to 4. Forexample, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5. In someembodiments, n is 6.

In some other of the foregoing embodiments of Formula (III), y and z areeach independently an integer ranging from 2 to 10. For example, in someembodiments, y and z are each independently an integer ranging from 4 to9 or from 4 to 6.

In some of the foregoing embodiments of Formula (III), R⁶ is H. In otherof the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments,R⁶ is OH.

In some embodiments of Formula (III), G³ is unsubstituted. In otherembodiments, G³ is substituted. In various different embodiments, G³ islinear C₁-C₂₄ alkylene or linear C₁-C₂₄ alkenylene.

In some other foregoing embodiments of Formula (III), R¹ or R², or both,is C₆-C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each,independently have the following structure:

wherein:R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂alkyl; anda is an integer from 2 to 12,wherein R^(7a), R^(7b) and a are each selected such that R¹ and R² eachindependently comprise from 6 to 20 carbon atoms. For example, in someembodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments of Formula (III), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of Formula (III), R¹ or R², or both, has one ofthe following structures:

In some of the foregoing embodiments of Formula (III), R³ is OH, CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl orethyl.

In various different embodiments, the cationic lipid of Formula (III)has one of the structures set forth in the table below.

Representative Compounds of Formula (III).

No. Structure III-1 

III-2 

III-3 

III-4 

III-5 

III-6 

III-7 

III-8 

III-9 

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

III-20

III-21

III-22

III-23

III-24

III-25

III-26

III-27

III-28

III-29

III-30

III-31

III-32

III-33

III-34

III-35

III-36

In some embodiments, the LNP comprises a lipid of Formula (III), RNA, aneutral lipid, a steroid and a pegylated lipid. In some embodiments, thelipid of Formula (III) is compound III-3. In some embodiments, theneutral lipid is DSPC. In some embodiments, the steroid is cholesterol.In some embodiments, the pegylated lipid is ALC-0159.

In some embodiments, the cationic lipid is present in the LNP in anamount from about 40 to about 50 mole percent. In one embodiment, theneutral lipid is present in the LNP in an amount from about 5 to about15 mole percent. In one embodiment, the steroid is present in the LNP inan amount from about 35 to about 45 mole percent. In one embodiment, thepegylated lipid is present in the LNP in an amount from about 1 to about10 mole percent. In some embodiments, the LNP comprises compound III-3in an amount from about 40 to about 50 mole percent, DSPC in an amountfrom about 5 to about 15 mole percent, cholesterol in an amount fromabout 35 to about 45 mole percent, and ALC-0159 in an amount from about1 to about 10 mole percent.

In some embodiments, the LNP comprises compound III-3 in an amount ofabout 47.5 mole percent, DSPC in an amount of about 10 mole percent,cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in anamount of about 1.8 mole percent.

In various different embodiments, the cationic lipid has one of thestructures set forth in the table below.

No. Structure A

B

C

D

E

F

In some embodiments, the LNP comprises a cationic lipid shown in theabove table, e.g., a cationic lipid of Formula (B) or Formula (D), inparticular a cationic lipid of Formula (D), RNA, a neutral lipid, asteroid and a pegylated lipid. In some embodiments, the neutral lipid isDSPC. In some embodiments, the steroid is cholesterol. In someembodiments, the pegylated lipid is DMG-PEG 2000.

In one embodiment, the LNP comprises a cationic lipid that is anionizable lipid-like material (lipidoid). In one embodiment, thecationic lipid has the following structure:

The N/P value is preferably at least about 4. In some embodiments, theN/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. Inone embodiment, the N/P value is about 6.

LNP described herein may have an average diameter that in one embodimentranges from about 30 nm to about 200 nm, or from about 60 nm to about120 nm.

RNA Targeting

Some aspects of the disclosure involve the targeted delivery of the RNAdisclosed herein (e.g., RNA encoding vaccine antigens and/orimmunostimulants).

In one embodiment, the disclosure involves targeting lung. Targetinglung is in particular preferred if the RNA administered is RNA encodingvaccine antigen. RNA may be delivered to lung, for example, byadministering the RNA which may be formulated as particles as describedherein, e.g., lipid particles, by inhalation.

In one embodiment, the disclosure involves targeting the lymphaticsystem, in particular secondary lymphoid organs, more specificallyspleen. Targeting the lymphatic system, in particular secondary lymphoidorgans, more specifically spleen is in particular preferred if the RNAadministered is RNA encoding vaccine antigen.

In one embodiment, the target cell is a spleen cell. In one embodiment,the target cell is an antigen presenting cell such as a professionalantigen presenting cell in the spleen. In one embodiment, the targetcell is a dendritic cell in the spleen.

The “lymphatic system” is part of the circulatory system and animportant part of the immune system, comprising a network of lymphaticvessels that carry lymph. The lymphatic system consists of lymphaticorgans, a conducting network of lymphatic vessels, and the circulatinglymph. The primary or central lymphoid organs generate lymphocytes fromimmature progenitor cells. The thymus and the bone marrow constitute theprimary lymphoid organs. Secondary or peripheral lymphoid organs, whichinclude lymph nodes and the spleen, maintain mature naïve lymphocytesand initiate an adaptive immune response.

RNA may be delivered to spleen by so-called lipoplex formulations, inwhich the RNA is bound to liposomes comprising a cationic lipid andoptionally an additional or helper lipid to form injectable nanoparticleformulations. The liposomes may be obtained by injecting a solution ofthe lipids in ethanol into water or a suitable aqueous phase. RNAlipoplex particles may be prepared by mixing the liposomes with RNA.Spleen targeting RNA lipoplex particles are described in WO 2013/143683,herein incorporated by reference. It has been found that RNA lipoplexparticles having a net negative charge may be used to preferentiallytarget spleen tissue or spleen cells such as antigen-presenting cells,in particular dendritic cells. Accordingly, following administration ofthe RNA lipoplex particles, RNA accumulation and/or RNA expression inthe spleen occurs. Thus, RNA lipoplex particles of the disclosure may beused for expressing RNA in the spleen. In an embodiment, afteradministration of the RNA lipoplex particles, no or essentially no RNAaccumulation and/or RNA expression in the lung and/or liver occurs. Inone embodiment, after administration of the RNA lipoplex particles, RNAaccumulation and/or RNA expression in antigen presenting cells, such asprofessional antigen presenting cells in the spleen occurs. Thus, RNAlipoplex particles of the disclosure may be used for expressing RNA insuch antigen presenting cells. In one embodiment, the antigen presentingcells are dendritic cells and/or macrophages.

The electric charge of the RNA lipoplex particles of the presentdisclosure is the sum of the electric charges present in the at leastone cationic lipid and the electric charges present in the RNA. Thecharge ratio is the ratio of the positive charges present in the atleast one cationic lipid to the negative charges present in the RNA. Thecharge ratio of the positive charges present in the at least onecationic lipid to the negative charges present in the RNA is calculatedby the following equation: charge ratio=[(cationic lipid concentration(mol))*(the total number of positive charges in the cationiclipid)]/[(RNA concentration (mol))*(the total number of negative chargesin RNA)].

The spleen targeting RNA lipoplex particles described herein atphysiological pH preferably have a net negative charge such as a chargeratio of positive charges to negative charges from about 1.9:2 to about1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. Inspecific embodiments, the charge ratio of positive charges to negativecharges in the RNA lipoplex particles at physiological pH is about1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0,about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about1:2.0.

Immunostimulants may be provided to a subject by administering to thesubject RNA encoding an immunostimulant in a formulation forpreferential delivery of RNA to liver or liver tissue. The delivery ofRNA to such target organ or tissue is preferred, in particular, if it isdesired to express large amounts of the immunostimulant and/or ifsystemic presence of the immunostimulant, in particular in significantamounts, is desired or required.

RNA delivery systems have an inherent preference to the liver. Thispertains to lipid-based particles, cationic and neutral nanoparticles,in particular lipid nanoparticles such as liposomes, nanomicelles andlipophilic ligands in bioconjugates. Liver accumulation is caused by thediscontinuous nature of the hepatic vasculature or the lipid metabolism(liposomes and lipid or cholesterol conjugates).

For in vivo delivery of RNA to the liver, a drug delivery system may beused to transport the RNA into the liver by preventing its degradation.For example, polyplex nanomicelles consisting of a poly(ethylene glycol)(PEG)-coated surface and an mRNA-containing core is a useful systembecause the nanomicelles provide excellent in vivo stability of the RNA,under physiological conditions. Furthermore, the stealth propertyprovided by the polyplex nanomicelle surface, composed of dense PEGpalisades, effectively evades host immune defenses.

Examples of suitable immunostimulants for targeting liver are cytokinesinvolved in T cell proliferation and/or maintenance. Examples ofsuitable cytokines include IL2 or IL7, fragments and variants thereof,and fusion proteins of these cytokines, fragments and variants, such asextended-PK cytokines.

In another embodiment, RNA encoding an immunostimulant may beadministered in a formulation for preferential delivery of RNA to thelymphatic system, in particular secondary lymphoid organs, morespecifically spleen. The delivery of an immunostimulant to such targettissue is preferred, in particular, if presence of the immunostimulantin this organ or tissue is desired (e.g., for inducing an immuneresponse, in particular in case immunostimulants such as cytokines arerequired during T-cell priming or for activation of resident immunecells), while it is not desired that the immunostimulant is presentsystemically, in particular in significant amounts (e.g., because theimmunostimulant has systemic toxicity).

Examples of suitable immunostimulants are cytokines involved in T cellpriming. Examples of suitable cytokines include IL12, IL15, IFN-α, orIFN-β, fragments and variants thereof, and fusion proteins of thesecytokines, fragments and variants, such as extended-PK cytokines.

Immunostimulants

In one embodiment, the RNA encoding vaccine antigen may benon-immunogenic. In this and other embodiments, the RNA encoding vaccineantigen may be co-administered with an immunostimulant or RNA encodingan immunostimulant. The methods and agents described herein areparticularly effective if the immunostimulant is attached to apharmacokinetic modifying group (hereafter referred to as“extended-pharmacokinetic (PK)” immunostimulant). The methods and agentsdescribed herein are particularly effective if the immunostimulant isadministered in the form of RNA encoding an immunostimulant. In oneembodiment, said RNA is targeted to the liver for systemic availability.Liver cells can be efficiently transfected and are able to produce largeamounts of protein.

An “immunostimulant” is any substance that stimulates the immune systemby inducing activation or increasing activity of any of the immunesystem's components, in particular immune effector cells. Theimmunostimulant may be pro-inflammatory.

According to one aspect, the immunostimulant is a cytokine or a variantthereof. Examples of cytokines include interferons, such asinterferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins, suchas IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such asM-CSF and GM-CSF, and tumor necrosis factor. According to anotheraspect, the immunostimulant includes an adjuvant-type immunostimulatoryagent such as APC Toll-like Receptor agonists or costimulatory/celladhesion membrane proteins. Examples of Toll-like Receptor agonistsinclude costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.

Cytokines are a category of small proteins (˜5-20 kDa) that areimportant in cell signaling. Their release has an effect on the behaviorof cells around them. Cytokines are involved in autocrine signaling,paracrine signaling and endocrine signaling as immunomodulating agents.Cytokines include chemokines, interferons, interleukins, lymphokines,and tumour necrosis factors but generally not hormones or growth factors(despite some overlap in the terminology). Cytokines are produced by abroad range of cells, including immune cells like macrophages, Blymphocytes, T lymphocytes and mast cells, as well as endothelial cells,fibroblasts, and various stromal cells. A given cytokine may be producedby more than one type of cell. Cytokines act through receptors, and areespecially important in the immune system; cytokines modulate thebalance between humoral and cell-based immune responses, and theyregulate the maturation, growth, and responsiveness of particular cellpopulations. Some cytokines enhance or inhibit the action of othercytokines in complex ways.

According to the disclosure, a cytokine may be a naturally occurringcytokine or a functional fragment or variant thereof. A cytokine may behuman cytokine and may be derived from any vertebrate, especially anymammal. One particularly preferred cytokine is interferon-α.

Interferons

Interferons (IFNs) are a group of signaling proteins made and releasedby host cells in response to the presence of several pathogens, such asviruses, bacteria, parasites, and also tumor cells. In a typicalscenario, a virus-infected cell will release interferons causing nearbycells to heighten their anti-viral defenses.

Based on the type of receptor through which they signal, interferons aretypically divided among three classes: type I interferon, type IIinterferon, and type 11 interferon.

All type I interferons bind to a specific cell surface receptor complexknown as the IFN-α/3 receptor (IFNAR) that consists of IFNAR1 and IFNAR2chains.

The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ andIFNω. In general, type I interferons are produced when the bodyrecognizes a virus that has invaded it. They are produced by fibroblastsand monocytes. Once released, type I interferons bind to specificreceptors on target cells, which leads to expression of proteins thatwill prevent the virus from producing and replicating its RNA and DNA.

The IFNα proteins are produced mainly by plasmacytoid dendritic cells(pDCs). They are mainly involved in innate immunity against viralinfection. The genes responsible for their synthesis come in 13 subtypesthat are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10,IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found togetherin a cluster on chromosome 9.

The IFNβ proteins are produced in large quantities by fibroblasts. Theyhave antiviral activity that is involved mainly in innate immuneresponse. Two types of IFNβ have been described, IFNβ1 and IFNβ3. Thenatural and recombinant forms of IFNβ1 have antiviral, antibacterial,and anticancer properties.

Type II interferon (IFNγ in humans) is also known as immune interferonand is activated by IL12. Furthermore, type II interferons are releasedby cytotoxic T cells and T helper cells. Type III interferons signalthrough a receptor complex consisting of IL10R2 (also called CRF2-4) andIFNLR1 (also called CRF2-12). Although discovered more recently thantype I and type II IFNs, recent information demonstrates the importanceof type III IFNs in some types of virus or fungal infections.

In general, type I and II interferons are responsible for regulating andactivating the immune response.

According to the disclosure, a type I interferon is preferably IFNα orIFNβ, more preferably IFNα.

According to the disclosure, an interferon may be a naturally occurringinterferon or a functional fragment or variant thereof. An interferonmay be human interferon and may be derived from any vertebrate,especially any mammal.

Interleukins

Interleukins (ILs) are a group of cytokines (secreted proteins andsignal molecules) that can be divided into four major groups based ondistinguishing structural features. However, their amino acid sequencesimilarity is rather weak (typically 15-25% identity). The human genomeencodes more than 50 interleukins and related proteins.

According to the disclosure, an interleukin may be a naturally occurringinterleukin or a functional fragment or variant thereof. An interleukinmay be human interleukin and may be derived from any vertebrate,especially any mammal.

Extended-PK Group

Immunostimulant polypeptides described herein can be prepared as fusionor chimeric polypeptides that include an immunostimulant portion and aheterologous polypeptide (i.e., a polypeptide that is not animmunostimulant). The immunostimulant may be fused to an extended-PKgroup, which increases circulation half-life. Non-limiting examples ofextended-PK groups are described infra. It should be understood thatother PK groups that increase the circulation half-life ofimmunostimulants such as cytokines, or variants thereof, are alsoapplicable to the present disclosure. In certain embodiments, theextended-PK group is a serum albumin domain (e.g., mouse serum albumin,human serum albumin).

As used herein, the term “PK” is an acronym for “pharmacokinetic” andencompasses properties of a compound including, by way of example,absorption, distribution, metabolism, and elimination by a subject. Asused herein, an “extended-PK group” refers to a protein, peptide, ormoiety that increases the circulation half-life of a biologically activemolecule when fused to or administered together with the biologicallyactive molecule. Examples of an extended-PK group include serum albumin(e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof,transferrin and variants thereof, and human serum albumin (HSA) binders(as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).Other exemplary extended-PK groups are disclosed in Kontermann, ExpertOpin Biol Ther, 2016 July; 16(7):903-15 which is herein incorporated byreference in its entirety. As used herein, an “extended-PK”immunostimulant refers to an immunostimulant moiety in combination withan extended-PK group. In one embodiment, the extended-PK immunostimulantis a fusion protein in which an immunostimulant moiety is linked orfused to an extended-PK group.

In certain embodiments, the serum half-life of an extended-PKimmunostimulant is increased relative to the immunostimulant alone(i.e., the immunostimulant not fused to an extended-PK group). Incertain embodiments, the serum half-life of the extended-PKimmunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200,400, 600, 800, or 1000% longer relative to the serum half-life of theimmunostimulant alone. In certain embodiments, the serum half-life ofthe extended-PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold,3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold,27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serumhalf-life of the immunostimulant alone. In certain embodiments, theserum half-life of the extended-PK immunostimulant is at least 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours,130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

As used herein, “half-life” refers to the time taken for the serum orplasma concentration of a compound such as a peptide or protein toreduce by 50%, in vivo, for example due to degradation and/or clearanceor sequestration by natural mechanisms. An extended-PK immunostimulantsuitable for use herein is stabilized in vivo and its half-lifeincreased by, e.g., fusion to serum albumin (e.g., HSA or MSA), whichresist degradation and/or clearance or sequestration. The half-life canbe determined in any manner known per se, such as by pharmacokineticanalysis. Suitable techniques will be clear to the person skilled in theart, and may for example generally involve the steps of suitablyadministering a suitable dose of the amino acid sequence or compound toa subject; collecting blood samples or other samples from said subjectat regular intervals; determining the level or concentration of theamino acid sequence or compound in said blood sample; and calculating,from (a plot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound has been reduced by50% compared to the initial level upon dosing. Further details areprovided in, e.g., standard handbooks, such as Kenneth, A. et al.,Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and inPeters et al., Pharmacokinetic Analysis: A Practical Approach (1996).Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev.Edition, Marcel Dekker (1982).

In certain embodiments, the extended-PK group includes serum albumin, orfragments thereof or variants of the serum albumin or fragments thereof(all of which for the purpose of the present disclosure are comprised bythe term “albumin”). Polypeptides described herein may be fused toalbumin (or a fragment or variant thereof) to form albumin fusionproteins. Such albumin fusion proteins are described in U.S. PublicationNo. 20070048282.

As used herein, “albumin fusion protein” refers to a protein formed bythe fusion of at least one molecule of albumin (or a fragment or variantthereof) to at least one molecule of a protein such as a therapeuticprotein, in particular an immunostimulant. The albumin fusion proteinmay be generated by translation of a nucleic acid in which apolynucleotide encoding a therapeutic protein is joined in-frame with apolynucleotide encoding an albumin. The therapeutic protein and albumin,once part of the albumin fusion protein, may each be referred to as a“portion”, “region” or “moiety” of the albumin fusion protein (e.g., a“therapeutic protein portion” or an “albumin protein portion”). In ahighly preferred embodiment, an albumin fusion protein comprises atleast one molecule of a therapeutic protein (including, but not limitedto a mature form of the therapeutic protein) and at least one moleculeof albumin (including but not limited to a mature form of albumin). Inone embodiment, an albumin fusion protein is processed by a host cellsuch as a cell of the target organ for administered RNA, e.g. a livercell, and secreted into the circulation. Processing of the nascentalbumin fusion protein that occurs in the secretory pathways of the hostcell used for expression of the RNA may include, but is not limited tosignal peptide cleavage; formation of disulfide bonds; proper folding;addition and processing of carbohydrates (such as for example, N- andO-linked glycosylation); specific proteolytic cleavages; and/or assemblyinto multimeric proteins. An albumin fusion protein is preferablyencoded by RNA in a non-processed form which in particular has a signalpeptide at its N-terminus and following secretion by a cell ispreferably present in the processed form wherein in particular thesignal peptide has been cleaved off. In a most preferred embodiment, the“processed form of an albumin fusion protein” refers to an albuminfusion protein product which has undergone N-terminal signal peptidecleavage, herein also referred to as a “mature albumin fusion protein”.In preferred embodiments, albumin fusion proteins comprising atherapeutic protein have a higher plasma stability compared to theplasma stability of the same therapeutic protein when not fused toalbumin. Plasma stability typically refers to the time period betweenwhen the therapeutic protein is administered in vivo and carried intothe bloodstream and when the therapeutic protein is degraded and clearedfrom the bloodstream, into an organ, such as the kidney or liver, thatultimately clears the therapeutic protein from the body. Plasmastability is calculated in terms of the half-life of the therapeuticprotein in the bloodstream. The half-life of the therapeutic protein inthe bloodstream can be readily determined by common assays known in theart.

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments or variantsthereof especially the mature form of human albumin, or albumin fromother vertebrates or fragments thereof, or variants of these molecules.The albumin may be derived from any vertebrate, especially any mammal,for example human, cow, sheep, or pig. Non-mammalian albumins include,but are not limited to, hen and salmon. The albumin portion of thealbumin fusion protein may be from a different animal than thetherapeutic protein portion.

In certain embodiments, the albumin is human serum albumin (HSA), orfragments or variants thereof, such as those disclosed in U.S. Pat. No.5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

As used herein, a fragment of albumin sufficient to prolong thetherapeutic activity or plasma stability of the therapeutic proteinrefers to a fragment of albumin sufficient in length or structure tostabilize or prolong the therapeutic activity or plasma stability of theprotein so that the plasma stability of the therapeutic protein portionof the albumin fusion protein is prolonged or extended compared to theplasma stability in the non-fusion state.

The albumin portion of the albumin fusion proteins may comprise the fulllength of the albumin sequence, or may include one or more fragmentsthereof that are capable of stabilizing or prolonging the therapeuticactivity or plasma stability. Such fragments may be of 10 or more aminoacids in length or may include about 15, 20, 25, 30, 50, or morecontiguous amino acids from the albumin sequence or may include part orall of specific domains of albumin. For instance, one or more fragmentsof HSA spanning the first two immunoglobulin-like domains may be used.In a preferred embodiment, the HSA fragment is the mature form of HSA.

Generally speaking, an albumin fragment or variant will be at least 100amino acids long, preferably at least 150 amino acids long.

According to the disclosure, albumin may be naturally occurring albuminor a fragment or variant thereof. Albumin may be human albumin and maybe derived from any vertebrate, especially any mammal.

Preferably, the albumin fusion protein comprises albumin as theN-terminal portion, and a therapeutic protein as the C-terminal portion.Alternatively, an albumin fusion protein comprising albumin as theC-terminal portion, and a therapeutic protein as the N-terminal portionmay also be used. In other embodiments, the albumin fusion protein has atherapeutic protein fused to both the N-terminus and the C-terminus ofalbumin. In a preferred embodiment, the therapeutic proteins fused atthe N- and C-termini are the same therapeutic proteins. In anotherpreferred embodiment, the therapeutic proteins fused at the N- andC-termini are different therapeutic proteins. In one embodiment, thedifferent therapeutic proteins are both cytokines.

In one embodiment, the therapeutic protein(s) is (are) joined to thealbumin through (a) peptide linker(s). A linker peptide between thefused portions may provide greater physical separation between themoieties and thus maximize the accessibility of the therapeutic proteinportion, for instance, for binding to its cognate receptor. The linkerpeptide may consist of amino acids such that it is flexible or morerigid. The linker sequence may be cleavable by a protease or chemically.

As used herein, the term “Fc region” refers to the portion of a nativeimmunoglobulin formed by the respective Fc domains (or Fc moieties) ofits two heavy chains. As used herein, the term “Fc domain” refers to aportion or fragment of a single immunoglobulin (Ig) heavy chain whereinthe Fc domain does not comprise an Fv domain. In certain embodiments, anFc domain begins in the hinge region just upstream of the papaincleavage site and ends at the C-terminus of the antibody. Accordingly, acomplete Fc domain comprises at least a hinge domain, a CH2 domain, anda CH3 domain. In certain embodiments, an Fc domain comprises at leastone of: a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, orfragment thereof. In certain embodiments, an Fc domain comprises acomplete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3domain). In certain embodiments, an Fc domain comprises a hinge domain(or portion thereof) fused to a CH3 domain (or portion thereof). Incertain embodiments, an Fc domain comprises a CH2 domain (or portionthereof) fused to a CH3 domain (or portion thereof). In certainembodiments, an Fc domain consists of a CH3 domain or portion thereof.In certain embodiments, an Fc domain consists of a hinge domain (orportion thereof) and a CH3 domain (or portion thereof). In certainembodiments, an Fc domain consists of a CH2 domain (or portion thereof)and a CH3 domain. In certain embodiments, an Fc domain consists of ahinge domain (or portion thereof) and a CH2 domain (or portion thereof).In certain embodiments, an Fc domain lacks at least a portion of a CH2domain (e.g., all or part of a CH2 domain). An Fc domain hereingenerally refers to a polypeptide comprising all or part of the Fcdomain of an immunoglobulin heavy-chain. This includes, but is notlimited to, polypeptides comprising the entire CH1, hinge, CH2, and/orCH3 domains as well as fragments of such peptides comprising only, e.g.,the hinge, CH2, and CH3 domain. The Fc domain may be derived from animmunoglobulin of any species and/or any subtype, including, but notlimited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. The Fc domain encompasses native Fc and Fc variant molecules.As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain embodiments, the Fc domain hasreduced effector function (e.g., FcγR binding).

The Fc domains of a polypeptide described herein may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

In certain embodiments, an extended-PK group includes an Fc domain orfragments thereof or variants of the Fc domain or fragments thereof (allof which for the purpose of the present disclosure are comprised by theterm “Fc domain”). The Fc domain does not contain a variable region thatbinds to antigen. Fc domains suitable for use in the present disclosuremay be obtained from a number of different sources. In certainembodiments, an Fc domain is derived from a human immunoglobulin. Incertain embodiments, the Fc domain is from a human IgG1 constant region.It is understood, however, that the Fc domain may be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the Fc domain (or afragment or variant thereof) may be derived from any immunoglobulinclass, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulinisotype, including IgG1, IgG2, IgG3, and IgG4.

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragmentsor variants thereof) can be derived from these sequences using artrecognized techniques.

In certain embodiments, the extended-PK group is a serum albumin bindingprotein such as those described in US2005/0287153, US2007/0003549,US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, andWO2009/133208, which are herein incorporated by reference in theirentirety. In certain embodiments, the extended-PK group is transferrin,as disclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579, which are hereinincorporated by reference in their entirety. In certain embodiments, theextended-PK group is a serum immunoglobulin binding protein such asthose disclosed in US2007/0178082, US2014/0220017, and US2017/0145062,which are herein incorporated by reference in their entirety. In certainembodiments, the extended-PK group is a fibronectin (Fn)-based scaffolddomain protein that binds to serum albumin, such as those disclosed inUS2012/0094909, which is herein incorporated by reference in itsentirety. Methods of making fibronectin-based scaffold domain proteinsare also disclosed in US2012/0094909. A non-limiting example of aFn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that bindsto human serum albumin. In certain aspects, the extended-PKimmunostimulant, suitable for use according to the disclosure, canemploy one or more peptide linkers. As used herein, the term “peptidelinker” refers to a peptide or polypeptide sequence which connects twoor more domains (e.g., the extended-PK moiety and an immunostimulantmoiety) in a linear amino acid sequence of a polypeptide chain. Forexample, peptide linkers may be used to connect an immunostimulantmoiety to a HSA domain.

Linkers suitable for fusing the extended-PK group to e.g. animmunostimulant are well known in the art. Exemplary linkers includeglycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers,and proline-alanine polypeptide linkers. In certain embodiments, thelinker is a glycine-serine-polypeptide linker, i.e., a peptide thatconsists of glycine and serine residues.

In addition to, or in place of, the heterologous polypeptides describedabove, an immunostimulant polypeptide described herein can containsequences encoding a “marker” or “reporter”. Examples of marker orreporter genes include β-lactamase, chloramphenicol acetyltransferase(CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase,dihydrofolate reductase (DHFR), hygromycin-B-hosphotransferase (HPH),thymidine kinase (TK), β-galactosidase, and xanthine guaninephosphoribosyltransferase (XGPRT).

Pharmaceutical Compositions

The agents described herein may be administered in pharmaceuticalcompositions or medicaments and may be administered in the form of anysuitable pharmaceutical composition.

In one embodiment, the pharmaceutical composition described herein is animmunogenic composition for inducing an immune response againstcoronavirus in a subject. For example, in one embodiment, theimmunogenic composition is a vaccine.

In one embodiment of all aspects of the invention, the componentsdescribed herein such as RNA encoding a vaccine antigen may beadministered in a pharmaceutical composition which may comprise apharmaceutically acceptable carrier and may optionally comprise one ormore adjuvants, stabilizers etc. In one embodiment, the pharmaceuticalcomposition is for therapeutic or prophylactic treatments, e.g., for usein treating or preventing a coronavirus infection.

The term “pharmaceutical composition” relates to a formulationcomprising a therapeutically effective agent, preferably together withpharmaceutically acceptable carriers, diluents and/or excipients. Saidpharmaceutical composition is useful for treating, preventing, orreducing the severity of a disease or disorder by administration of saidpharmaceutical composition to a subject. A pharmaceutical composition isalso known in the art as a pharmaceutical formulation.

The pharmaceutical compositions of the present disclosure may compriseone or more adjuvants or may be administered with one or more adjuvants.The term “adjuvant” relates to a compound which prolongs, enhances oraccelerates an immune response. Adjuvants comprise a heterogeneous groupof compounds such as oil emulsions (e.g., Freund's adjuvants), mineralcompounds (such as alum), bacterial products (such as Bordetellapertussis toxin), or immune-stimulating complexes. Examples of adjuvantsinclude, without limitation, LPS, GP96, CpG oligodeoxynucleotides,growth factors, and cytokines, such as monokines, lymphokines,interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5,IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further knownadjuvants are aluminium hydroxide, Freund's adjuvant or oil such asMontanide® ISA51. Other suitable adjuvants for use in the presentdisclosure include lipopeptides, such as Pam3Cys.

The pharmaceutical compositions according to the present disclosure aregenerally applied in a “pharmaceutically effective amount” and in “apharmaceutically acceptable preparation”. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinteract with the action of the active component of the pharmaceuticalcomposition.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to the amount which achieves a desired reactionor a desired effect alone or together with further doses. In the case ofthe treatment of a particular disease, the desired reaction preferablyrelates to inhibition of the course of the disease. This comprisesslowing down the progress of the disease and, in particular,interrupting or reversing the progress of the disease. The desiredreaction in a treatment of a disease may also be delay of the onset or aprevention of the onset of said disease or said condition. An effectiveamount of the compositions described herein will depend on the conditionto be treated, the severeness of the disease, the individual parametersof the patient, including age, physiological condition, size and weight,the duration of treatment, the type of an accompanying therapy (ifpresent), the specific route of administration and similar factors.Accordingly, the doses administered of the compositions described hereinmay depend on various of such parameters. In the case that a reaction ina patient is insufficient with an initial dose, higher doses (oreffectively higher doses achieved by a different, more localized routeof administration) may be used.

The pharmaceutical compositions of the present disclosure may containsalts, buffers, preservatives, and optionally other therapeutic agents.In one embodiment, the pharmaceutical compositions of the presentdisclosure comprise one or more pharmaceutically acceptable carriers,diluents and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of thepresent disclosure include, without limitation, benzalkonium chloride,chlorobutanol, paraben and thimerosal. The term “excipient” as usedherein refers to a substance which may be present in a pharmaceuticalcomposition of the present disclosure but is not an active ingredient.Examples of excipients, include without limitation, carriers, binders,diluents, lubricants, thickeners, surface active agents, preservatives,stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term “diluent” relates a diluting and/or thinning agent. Moreover,the term “diluent” includes any one or more of fluid, liquid or solidsuspension and/or mixing media. Examples of suitable diluents includeethanol, glycerol and water.

The term “carrier” refers to a component which may be natural,synthetic, organic, inorganic in which the active component is combinedin order to facilitate, enhance or enable administration of thepharmaceutical composition. A carrier as used herein may be one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to subject. Suitablecarrier include, without limitation, sterile water, Ringer, Ringerlactate, sterile sodium chloride solution, isotonic saline, polyalkyleneglycols, hydrogenated naphthalenes and, in particular, biocompatiblelactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxy-propylene copolymers. In one embodiment, thepharmaceutical composition of the present disclosure includes isotonicsaline.

Pharmaceutically acceptable carriers, excipients or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents can be selected withregard to the intended route of administration and standardpharmaceutical practice.

In one embodiment, pharmaceutical compositions described herein may beadministered intravenously, intraarterially, subcutaneously,intradermally or intramuscularly. In certain embodiments, thepharmaceutical composition is formulated for local administration orsystemic administration. Systemic administration may include enteraladministration, which involves absorption through the gastrointestinaltract, or parenteral administration. As used herein, “parenteraladministration” refers to the administration in any manner other thanthrough the gastrointestinal tract, such as by intravenous injection. Ina preferred embodiment, the pharmaceutical composition is formulated forintramuscular administration. In another embodiment, the pharmaceuticalcomposition is formulated for systemic administration, e.g., forintravenous administration.

The term “co-administering” as used herein means a process wherebydifferent compounds or compositions (e.g., RNA encoding an antigen andRNA encoding an immunostimulant) are administered to the same patient.The different compounds or compositions may be administeredsimultaneously, at essentially the same time, or sequentially.

The pharmaceutical compositions and products described herein may beprovided as a frozen concentrate for solution for injection, e.g., at aconcentration of 0.50 mg/mL. In one embodiment, for preparation ofsolution for injection, a drug product is thawed and diluted withisotonic sodium chloride solution (e.g., 0.9% NaCl, saline), e.g., by aone-step dilution process. In some embodiments, bacteriostatic sodiumchloride solution (e.g., 0.9% NaCl, saline) cannot be used as a diluent.In some embodiments, a diluted drug product is an off-white suspension.The concentration of the final solution for injection varies dependingon the respective dose level to be administered.

In one embodiment, administration is performed within 6 h after begin ofpreparation due to the risk of microbial contamination and consideringthe multiple-dose approach of the preparation process. In oneembodiment, in this period of 6 h, two conditions are allowed: roomtemperature for preparation, handling and transfer as well as 2 to 8° C.for storage. Compositions described herein may be shipped and/or storedunder temperature-controlled conditions, e.g., temperature conditions ofabout 4-5° C. or below, about −20° C. or below, −70° C.±10° C. (e.g.,−80° C. to −60° C.), e.g., utilizing a cooling system (e.g., that may beor include dry ice) to maintain the desired temperature. In oneembodiment, compositions described herein are shipped intemperature-controlled thermal shippers. Such shippers may contain aGPS-enabled thermal sensor to track the location and temperature of eachshipment. The compositions can be stored by refilling with, e.g., dryice.

Treatments

The present invention provides methods and agents for inducing anadaptive immune response against coronavirus in a subject comprisingadministering an effective amount of a composition comprising RNAencoding a coronavirus vaccine antigen described herein.

In one embodiment, the methods and agents described herein provideimmunity in a subject to coronavirus, coronavirus infection, or to adisease or disorder associated with coronavirus. The present inventionthus provides methods and agents for treating or preventing theinfection, disease, or disorder associated with coronavirus.

In one embodiment, the methods and agents described herein areadministered to a subject having an infection, disease, or disorderassociated with coronavirus. In one embodiment, the methods and agentsdescribed herein are administered to a subject at risk for developingthe infection, disease, or disorder associated with coronavirus. Forexample, the methods and agents described herein may be administered toa subject who is at risk for being in contact with coronavirus. In oneembodiment, the methods and agents described herein are administered toa subject who lives in, traveled to, or is expected to travel to ageographic region in which coronavirus is prevalent. In one embodiment,the methods and agents described herein are administered to a subjectwho is in contact with or expected to be in contact with another personwho lives in, traveled to, or is expected to travel to a geographicregion in which coronavirus is prevalent. In one embodiment, the methodsand agents described herein are administered to a subject who hasknowingly been exposed to coronavirus through their occupation, or othercontact. In one embodiment, a coronavirus is SARS-CoV-2. In someembodiments, methods and agents described herein are administered to asubject with evidence of prior exposure to and/or infection withSARS-CoV-2 and/or an antigen or epitope thereof or cross-reactivetherewith. For example, in some embodiments, methods and agentsdescribed herein are administered to a subject in whom antibodies, Bcells, and/or T cells reactive with one or more epitopes of a SARS-CoV-2spike protein are detectable and/or have been detected.

For a composition to be useful as a vaccine, the composition must inducean immune response against the coronavirus antigen in a cell, tissue orsubject (e.g., a human). In some embodiments, the composition induces animmune response against the coronavirus antigen in a cell, tissue orsubject (e.g., a human). In some instances, the vaccine induces aprotective immune response in a mammal. The therapeutic compounds orcompositions of the invention may be administered prophylactically(i.e., to prevent a disease or disorder) or therapeutically (i.e., totreat a disease or disorder) to subjects suffering from, or at risk of(or susceptible to) developing a disease or disorder. Such subjects maybe identified using standard clinical methods. In the context of thepresent invention, prophylactic administration occurs prior to themanifestation of overt clinical symptoms of disease, such that a diseaseor disorder is prevented or alternatively delayed in its progression. Inthe context of the field of medicine, the term “prevent” encompasses anyactivity, which reduces the burden of mortality or morbidity fromdisease. Prevention can occur at primary, secondary and tertiaryprevention levels. While primary prevention avoids the development of adisease, secondary and tertiary levels of prevention encompassactivities aimed at preventing the progression of a disease and theemergence of symptoms as well as reducing the negative impact of analready established disease by restoring function and reducingdisease-related complications.

The present disclosure reports various characterization of providedcompositions (see, e.g., Example 2; see also Examples thereafter) andfurthermore establishes parameters for vaccines effective in humans.

In some embodiments, administration of an immunogenic composition orvaccine of the present invention may be performed by singleadministration or boosted by multiple administrations.

In some embodiments, an amount the RNA described herein from 0.1 μg to300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, such as about 1 μg, about 3μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg may beadministered per dose. In one embodiment, the invention envisionsadministration of a single dose. In one embodiment, the inventionenvisions administration of a priming dose followed by one or morebooster doses. The booster dose or the first booster dose may beadministered 7 to 28 days or 14 to 24 days following administration ofthe priming dose.

In some embodiments, an amount of the RNA described herein of 60 μg orlower, 50 μg or lower, 40 μg or lower, 30 μg or lower, 20 μg or lower,10 μg or lower, 5 μg or lower, 2.5 μg or lower, or 1 μg or lower may beadministered per dose.

In some embodiments, an amount of the RNA described herein of at least0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg,at least 4 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least30 μg, or at least 40 μg may be administered per dose.

In some embodiments, an amount of the RNA described herein of 0.25 μg to60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μgmay be administered per dose.

In one embodiment, an amount of the RNA described herein of about 30 μgis administered per dose. In one embodiment, at least two of such dosesare administered. For example, a second dose may be administered about21 days following administration of the first dose.

In some embodiments, the efficacy of the RNA vaccine described herein(e.g., administered in two doses, wherein a second dose may beadministered about 21 days following administration of the first dose,and administered, for example, in an amount of about 30 μg per dose) isat least 70%, at least 80%, at least 90, or at least 95% beginning 7days after administration of the second dose (e.g., beginning 28 daysafter administration of the first dose if a second dose is administered21 days following administration of the first dose). In someembodiments, such efficacy is observed in populations of age of at least50, at least 55, at least 60, at least 65, at least 70, or older. Insome embodiments, the efficacy of the RNA vaccine described herein(e.g., administered in two doses, wherein a second dose may beadministered about 21 days following administration of the first dose,and administered, for example, in an amount of about 30 μg per dose)beginning 7 days after administration of the second dose (e.g.,beginning 28 days after administration of the first dose if a seconddose is administered 21 days following administration of the first dose)in populations of age of at least 65, such as 65 to 80, 65 to 75, or 65to 70, is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, or at least 95%. Such efficacy may be observed over timeperiods of up to 1 month, 2 months, 3 months, 6 months or even longer.

In one embodiment, vaccine efficacy is defined as the percent reductionin the number of subjects with evidence of infection (vaccinatedsubjects vs. non-vaccinated subjects).

In one embodiment, efficacy is assessed through surveillance forpotential cases of COVID-19. If, at any time, a patient develops acuterespiratory illness, for the purposes herein, the patient can beconsidered to potentially have COVID-19 illness. The assessments caninclude a nasal (midturbinate) swab, which may be tested using a reversetranscription-polymerase chain reaction (RT-PCR) test to detectSARS-CoV-2. In addition, clinical information and results from localstandard-of-care tests can be assessed.

In some embodiments, efficacy assessments may utilize a definition ofSARS-CoV-2-related cases wherein:

-   -   Confirmed COVID-19: presence of at least 1 of the following        symptoms and SARS-CoV-2 NAAT (nucleic acid amplification-based        test) positive during, or within 4 days before or after, the        symptomatic period: fever; new or increased cough; new or        increased shortness of breath; chills; new or increased muscle        pain; new loss of taste or smell; sore throat; diarrhea;        vomiting.

Alternatively or additionally, in some embodiments, efficacy assessmentsmay utilize a definition of SARS-CoV-2-related cases wherein one or moreof the following additional symptoms defined by the CDC can beconsidered: fatigue; headache; nasal congestion or runny nose; nausea.

In some embodiments, efficacy assessments may utilize a definition ofSARS-CoV-2-related severe cases

-   -   Confirmed severe COVID-19: confirmed COVID-19 and presence of at        least 1 of the following: clinical signs at rest indicative of        severe systemic illness (e.g., RR≥30 breaths per minute, HR≥125        beats per minute, SpO₂≤93% on room air at sea level, or        PaO₂/FiO₂<300 mm Hg); respiratory failure (which can be defined        as needing high-flow oxygen, noninvasive ventilation, mechanical        ventilation, or ECMO); evidence of shock (e.g., SBP<90 mm Hg,        DBP<60 mm Hg, or requiring vasopressors); significant acute        renal, hepatic, or neurologic dysfunction; admission to an ICU;        death.

Alternatively or additionally, in some embodiments a serologicaldefinition can be used for patients without clinical presentation ofCOVID-19: e.g., confirmed seroconversion to SARS-CoV-2 without confirmedCOVID-19: e.g., positive N-binding antibody result in a patient with aprior negative N-binding antibody result.

In some embodiments, any or all of the following assays can be performedon serum samples: SARS-CoV-2 neutralization assay; S1-binding IgG levelassay; RBD-binding IgG level assay; N-binding antibody assay.

In one embodiment, methods and agents described herein are administeredto a paediatric population. In various embodiments, the paediatricpopulation comprises or consists of subjects under 18 years, e.g., 5 toless than 18 years of age, 12 to less than 18 years of age, 16 to lessthan 18 years of age, 12 to less than 16 years of age, or 5 to less than12 years of age. In various embodiments, the paediatric populationcomprises or consists of subjects under 5 years, e.g., 2 to less than 5years of age, 12 to less than 24 months of age, 7 to less than 12 monthsof age, or less than 6 months of age.

In one embodiment, the paediatric population comprises or consists ofsubjects 12 to less than 18 years of age including subjects 16 to lessthan 18 years of age and/or subjects 12 to less than 16 years of age. Inthis embodiment, treatments may comprise 2 vaccinations 21 days apart,wherein, in one embodiment, the vaccine is administered in an amount of30 μg RNA per dose, e.g., by intramuscular administration.

In one embodiment, the paediatric population comprises or consists ofsubjects 5 to less than 18 years of age including subjects 12 to lessthan 18 years of age and/or subjects 5 to less than 12 years of age. Inthis embodiment, treatments may comprise 2 vaccinations 21 days apart,wherein, in various embodiments, the vaccine is administered in anamount of 10 μg, 20 μg, or 30 μg RNA per dose, e.g., by intramuscularadministration.

In one embodiment, the paediatric population comprises or consists ofsubjects less than 5 years of age including subjects 2 to less than 5years of age, subjects 12 to less than 24 months of age, subjects 7 toless than 12 months of age, subjects 6 to less than 12 months of ageand/or subjects less than 6 months of age. In this embodiment,treatments may comprise 2 vaccinations, e.g., 21 to 42 days apart, e.g.,21 days apart, wherein, in various embodiments, the vaccine isadministered in an amount of 10 μg, 20 g, or 30 μg RNA per dose, e.g.,by intramuscular administration.

In some embodiments, efficacy for mRNA compositions described inpediatric populations (e.g., described herein) may be assessed byvarious metrics described herein (including, e.g., but not limited toCOVID-19 incidence per 1000 person-years in subjects with no serologicalor virological evidence of past SARS-CoV-2 infection; geometric meanratio (GMR) of SARS CoV-2 neutralizing titers measured, e.g., 7 daysafter a second dose; etc.)

In some embodiments, pediatric populations described herein (e.g., from12 to less than 16 years of age) may be monitored for occurrence ofmultisystem inflammatory syndrome (MIS) (e.g., inflammation in differentbody parts such as, e.g., heart, lung, kidneys, brain, skin, eyes,and/or gastrointestinal organs), after administration of an RNAcomposition (e.g., mRNA) described herein. Exemplary symponts of MIS inchildren may include, but are not limited to fever, abdominal pain,vomiting, diarrhea, neck pain, rash, bloodshot eyes, feeling extratried, and combinations thereof.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3),BNT162b2 (RBP020.1 or RBP020.2). In one embodiment, RNA administered asdescribed above is nucleoside modified messenger RNA (modRNA) describedherein as RBP020.2.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 21, a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 5, or an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of SEQ ID NO: 5. In one embodiment, RNAadministered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 5.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 19, or 20, a nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 7, or an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment,RNA administered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or20; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 20, a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 7, or an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of SEQ ID NO: 7. In one embodiment, RNAadministered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA administered is nucleoside modified messenger RNA(modRNA), (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof SEQ ID NO: 7, and is administered in an amount of about 30 μg perdose. In one embodiment, at least two of such doses are administered.For example, a second dose may be administered about 21 days followingadministration of the first dose.

In some embodiments, populations to be treated with RNA described hereincomprise, essentially consist of, or consist of subjects of age of atleast 50, at least 55, at least 60, or at least 65. In some embodiments,populations to be treated with RNA described herein comprise,essentially consist of, or consist of subjects of age of between 55 to90, 60 to 85, or 65 to 85.

In some embodiments, the period of time between the doses administeredis at least 7 days, at least 14 days, or at least 21 days. In someembodiments, the period of time between the doses administered isbetween 7 days and 28 days such as between 14 days and 23 days. In someembodiments, no more than 5 doses, no more than 4 doses, or no more than3 doses of the RNA described herein may be administered to a subject.

In some embodiments, the methods and agents described herein areadministered (in a regimen, e.g., at a dose, frequency of doses and/ornumber of doses) such that adverse events (AE), i.e., any unwantedmedical occurrence in a patient, e.g., any unfavourable and unintendedsign, symptom, or disease associated with the use of a medicinalproduct, whether or not related to the medicinal product, are mild ormoderate in intensity. In some embodiments, the methods and agentsdescribed herein are administered such that adverse events (AE) can bemanaged with interventions such as treatment with, e.g., paracetamol orother drugs that provide analgesic, antipyretic (fever-reducing) and/oranti-inflammatory effects, e.g., nonsteroidal anti-inflammatory drugs(NSAIDs), e.g., aspirin, ibuprofen, and naproxen. Paracetamol or“acetaminophen” which is not classified as a NSAID exerts weakanti-inflammatory effects and can be administered as analgesic accordingto the invention.

In some embodiments, the methods and agents described herein provide aneutralizing effect in a subject to coronavirus, coronavirus infection,or to a disease or disorder associated with coronavirus.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response that blocks orneutralizes coronavirus in the subject. In some embodiments, the methodsand agents described herein following administration to a subject inducethe generation of antibodies such as IgG antibodies that block orneutralize coronavirus in the subject. In some embodiments, the methodsand agents described herein following administration to a subject inducean immune response that blocks or neutralizes coronavirus S proteinbinding to ACE2 in the subject. In some embodiments, the methods andagents described herein following administration to a subject induce thegeneration of antibodies that block or neutralize coronavirus S proteinbinding to ACE2 in the subject.

In some embodiments, the methods and agents described herein followingadministration to a subject induce geometric mean concentrations (GMCs)of RBD domain-binding antibodies such as IgG antibodies of at least 500U/ml, 1000 U/ml, 2000 U/ml, 3000 U/ml, 4000 U/ml, 5000 U/ml, 10000 U/ml,15000 U/ml, 20000 U/ml, 25000 U/ml, 30000 U/ml or even higher. In someembodiments, the elevated GMCs of RBD domain-binding antibodies persistfor at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12months or even longer. In some embodiments, the methods and agentsdescribed herein following administration to a subject induce geometricmean titers (GMTs) of neutralizing antibodies such as IgG antibodies ofat least 100 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 1000 U/ml,1500 U/ml, or even higher. In some embodiments, the elevated GMTs ofneutralizing antibodies persist for at least 14 days, 21 days, 28 days,1 month, 3 months, 6 months, 12 months or even longer.

As used herein, the term “neutralization” refers to an event in whichbinding agents such as antibodies bind to a biological active site of avirus such as a receptor binding protein, thereby inhibiting the viralinfection of cells. As used herein, the term “neutralization” withrespect to coronavirus, in particular coronavirus S protein, refers toan event in which binding agents such as antibodies bind to the RBDdomain of the S protein, thereby inhibiting the viral infection ofcells. In particular, the term “neutralization” refers to an event inwhich binding agents eliminate or significantly reduce virulence (e.g.ability of infecting cells) of viruses of interest.

The type of immune response generated in response to an antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. Immune responses can be broadly dividedinto two types: Th1 and Th2. Th1 immune activation is optimized forintracellular infections such as viruses, whereas Th2 immune responsesare optimized for humoral (antibody) responses. Th1 cells produceinterleukin 2 (IL-2), tumor necrosis factor (TNFα) and interferon gamma(IFNγ). Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Th1immune activation is the most highly desired in many clinicalsituations. Vaccine compositions specialized in eliciting Th2 or humoralimmune responses are generally not effective against most viraldiseases.

In some embodiments, the methods and agents described herein followingadministration to a subject induce or promote a Th1-mediated immuneresponse in the subject. In some embodiments, the methods and agentsdescribed herein following administration to a subject induce or promotea cytokine profile that is typical for a Th1-mediated immune response inthe subject. In some embodiments, the methods and agents describedherein following administration to a subject induce or promote theproduction of interleukin 2 (IL-2), tumor necrosis factor (TNFα) and/orinterferon gamma (IFNγ) in the subject. In some embodiments, the methodsand agents described herein following administration to a subject induceor promote the production of interleukin 2 (IL-2) and interferon gamma(IFNγ) in the subject. In some embodiments, the methods and agentsdescribed herein following administration to a subject do not induce orpromote a Th2-mediated immune response in the subject, or induce orpromote a Th2-mediated immune response in the subject to a significantlower extent compared to the induction or promotion of a Th1-mediatedimmune response. In some embodiments, the methods and agents describedherein following administration to a subject do not induce or promote acytokine profile that is typical for a Th2-mediated immune response inthe subject, or induce or promote a cytokine profile that is typical fora Th2-mediated immune response in the subject to a significant lowerextent compared to the induction or promotion of a cytokine profile thatis typical for a Th1-mediated immune response. In some embodiments, themethods and agents described herein following administration to asubject do not induce or promote the production of IL-4, IL-5, IL-6,IL-9, IL-10 and/or IL-13, or induce or promote the production of IL-4,IL-5, IL-6, IL-9, IL-10 and/or IL-13 in the subject to a significantlower extent compared to the induction or promotion of interleukin 2(IL-2), tumor necrosis factor (TNFα) and/or interferon gamma (IFNγ) inthe subject. In some embodiments, the methods and agents describedherein following administration to a subject do not induce or promotethe production of IL-4, or induce or promote the production of IL-4 inthe subject to a significant lower extent compared to the induction orpromotion of interleukin 2 (IL-2) and interferon gamma (IFNγ) in thesubject. In some embodiments, the methods and agents described hereinfollowing administration to a subject induce an antibody response, inparticular a neutralizing antibody response, in the subject that targetsa panel of different S protein variants such as SARS-CoV-2 S proteinvariants, in particular naturally occurring S protein variants. In someembodiments, the panel of different S protein variants comprises atleast 5, at least 10, at least 15, or even more S protein variants. Insome embodiments, such S protein variants comprise variants having aminoacid modifications in the RBD domain and/or variants having amino acidmodifications outside the RBD domain. In one embodiment, such S proteinvariant comprises SARS-CoV-2 S protein or a naturally occurring variantthereof wherein the amino acid corresponding to position 321 (Q) in SEQID NO: 1 is S. In one embodiment, such S protein variant comprisesSARS-CoV-2 S protein or a naturally occurring variant thereof whereinthe amino acid corresponding to position 321 (Q) in SEQ ID NO: 1 is L.In one embodiment, such S protein variant comprises SARS-CoV-2 S proteinor a naturally occurring variant thereof wherein the amino acidcorresponding to position 341 (V) in SEQ ID NO: 1 is I. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 348 (A) in SEQ ID NO: 1 is T. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 354 (N)in SEQ ID NO: 1 is D. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 359 (S) in SEQ ID NO: 1is N. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 367 (V) in SEQ ID NO: 1 is F. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 378 (K) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 378 (K)in SEQ ID NO: 1 is R. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 408 (R) in SEQ ID NO: 1is I. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 409 (Q) in SEQ ID NO: 1 is E. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 435 (A) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 439 (N)in SEQ ID NO: 1 is K. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 458 (K) in SEQ ID NO: 1is R. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 472 (I) in SEQ ID NO: 1 is V. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 476 (G) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 477 (S)in SEQ ID NO: 1 is N. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 483 (V) in SEQ ID NO: 1is A. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 508 (Y) in SEQ ID NO: 1 is H. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 519 (H) in SEQ ID NO: 1 is P. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 614 (D)in SEQ ID NO: 1 is G.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 501 (N) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

Said S protein variant comprising a mutation at a position correspondingto position 501 (N) in SEQ ID NO: 1 may comprise one or more furthermutations. Such one or more further mutations may be selected frommutations at positions corresponding to the following positions in SEQID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T),982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R),417 (K), 242 (L), 243 (A), and 244 (L). In one embodiment, the aminoacid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G. In one embodiment, the amino acid corresponding toposition 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acidcorresponding to position 701 (A) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 18 (L) in SEQ IDNO: 1 is F. In one embodiment, the amino acid corresponding to position246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 242 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1. In some embodiments, themethods and agents described herein following administration to asubject induce an antibody response, in particular a neutralizingantibody response, in the subject that targets a S protein variant suchas SARS-CoV-2 S protein variant, in particular naturally occurring Sprotein variant comprising a deletion at a position corresponding topositions 69 (H) and 70 (V) in SEQ ID NO: 1.

In some embodiments, a S protein variant comprising a deletion at aposition corresponding to positions 69 (H) and 70 (V) in SEQ ID NO: 1may comprise one or more further mutations. Such one or more furthermutations may be selected from mutations at positions corresponding tothe following positions in SEQ ID NO: 1: 144 (Y), 501 (N), 570 (A), 614(D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701(A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692(I), 1147 (S), and 1229 (M). In one embodiment, the amino acidcorresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acidcorresponding to position 614 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 681 (P) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “Cluster 5”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, Y453F, I692V,M1229I, and optionally S1147L.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 614 (D) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 614 (D) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A),681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A),18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I),1147 (S), and 1229 (M). In one embodiment, the amino acid correspondingto position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted.In one embodiment, the amino acid corresponding to position 144 (Y) inSEQ ID NO: 1 is deleted. In one embodiment, the amino acid correspondingto position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the aminoacid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 681 (P) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,D614G and A701V, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y and the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1may comprise one or more further mutations. Such one or more furthermutations may be selected from mutations at positions corresponding tothe following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570(A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701(A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692(I), 1147 (S), and 1229 (M). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 681 (P) in SEQID NO: 1 is H. In one embodiment, the amino acid corresponding toposition 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,D614G and A701V, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 484 (E) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 484 (E) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A),614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A),18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I),1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H),1027 (T), and 1176 (V). In one embodiment, the amino acid correspondingto position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted.In one embodiment, the amino acid corresponding to position 144 (Y) inSEQ ID NO: 1 is deleted. In one embodiment, the amino acid correspondingto position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the aminoacid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 614 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 20 (T) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 138 (D) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 190 (R) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acidcorresponding to position 655 (H) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 1027 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.28”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y and the amino acid corresponding to position 484 (E) in SEQID NO: 1 is K.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1may comprise one or more further mutations. Such one or more furthermutations may be selected from mutations at positions corresponding tothe following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570(A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701(A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692(I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655(H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G. In one embodiment, the amino acid corresponding toposition 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 20 (T) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 138 (D) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 190 (R) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acidcorresponding to position 655 (H) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 1027 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1. In some embodiments, themethods and agents described herein following administration to asubject induce an antibody response, in particular a neutralizingantibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N), 484 (E) and 614 (D) in SEQ ID NO: 1.In one embodiment, the amino acid corresponding to position 501 (N) inSEQ ID NO: 1 is Y, the amino acid corresponding to position 484 (E) inSEQ ID NO: 1 is K and the amino acid corresponding to position 614 (D)in SEQ ID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N), 484 (E) and 614 (D) in SEQID NO: 1 may comprise one or more further mutations. Such one or morefurther mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1: 69 (H), 70(V), 144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215(D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453(Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417(K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 681 (P) in SEQID NO: 1 is H. In one embodiment, the amino acid corresponding toposition 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 701 (A) in SEQ IDNO: 1 is V. In one embodiment, the amino acid corresponding to position18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 246 (R) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acidcorresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 244 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 692 (I) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 1147 (S) in SEQ IDNO: 1 is L. In one embodiment, the amino acid corresponding to position1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 20 (T) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 26 (P) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 190 (R) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is T. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and D614G, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a deletion at a positioncorresponding to positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1.

In some embodiments, a S protein variant comprising a deletion at aposition corresponding to positions 242 (L), 243 (A) and 244 (L) in SEQID NO: 1 may comprise one or more further mutations. Such one or morefurther mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1: 69 (H), 70(V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118(D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 453(Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417(K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. Inone embodiment, the amino acid corresponding to position 570 (A) in SEQID NO: 1 is D. In one embodiment, the amino acid corresponding toposition 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 681 (P) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 716 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 1118 (D) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 80 (D) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 484 (E) in SEQ ID NO: 1 is K. In oneembodiment, the amino acid corresponding to position 701 (A) in SEQ IDNO: 1 is V. In one embodiment, the amino acid corresponding to position18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 246 (R) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 692 (I) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 1147 (S) in SEQ IDNO: 1 is L. In one embodiment, the amino acid corresponding to position1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 20 (T) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 26 (P) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 190 (R) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is T. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V and deletion 242-244, and optionally: L18F, R246I, and K417N. SaidS protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1. In some embodiments, themethods and agents described herein following administration to asubject induce an antibody response, in particular a neutralizingantibody response, in the subject that targets a S protein variant suchas SARS-CoV-2 S protein variant, in particular naturally occurring Sprotein variant comprising a mutation at a position corresponding toposition 417 (K) in SEQ ID NO: 1. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is T.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 417 (K) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A),614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E),701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I),1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T),and 1176 (V). In one embodiment, the amino acid corresponding toposition 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the aminoacid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 144 (Y) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 614 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acidcorresponding to position 701 (A) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 18 (L) in SEQ IDNO: 1 is F. In one embodiment, the amino acid corresponding to position246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 20 (T) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 138 (D) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 190 (R) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F. In some embodiments, the methods and agents described hereinfollowing administration to a subject induce an antibody response, inparticular a neutralizing antibody response, in the subject that targets501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and K417N, and optionally: L18F, R246I, and deletion 242-244.Said S protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 417 (K) and 484 (E) and/or 501 (N) in SEQ IDNO: 1. In one embodiment, the amino acid corresponding to position 417(K) in SEQ ID NO: 1 is N, and the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K and/or the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is T, and the aminoacid corresponding to position 484 (E) in SEQ ID NO: 1 is K and/or theamino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 417 (K) and 484 (E) and/or 501 (N)in SEQ ID NO: 1 may comprise one or more further mutations. Such one ormore further mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1: 69 (H), 70(V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80(D), 215 (D), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453(Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655(H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G. In one embodiment, the amino acid corresponding toposition 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acidcorresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 244 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 692 (I) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 1147 (S) in SEQ IDNO: 1 is L. In one embodiment, the amino acid corresponding to position1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 20 (T) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 26 (P) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 190 (R) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 655 (H) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and K417N and optionally: L18F, R246I, and deletion 242-244. SaidS protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

The term “amino acid corresponding to position . . . ” as used hereinrefers to an amino acid position number corresponding to an amino acidposition number in SARS-CoV-2 S protein, in particular the amino acidsequence shown in SEQ ID NO: 1. Corresponding amino acid positions inother coronavirus S protein variants such as SARS-CoV-2 S proteinvariants may be found by alignment with SARS-CoV-2 S protein, inparticular the amino acid sequence shown in SEQ ID NO: 1. It isconsidered well-known in the art how to align a sequence or segment in asequence and thereby determine the corresponding position in a sequenceto an amino acid position according to the present invention. Standardsequence alignment programs such as ALIGN, ClustalW or similar,typically at default settings may be used.

In some embodiments, the panel of different S protein variants to whichan antibody response is targeted comprises at least 5, at least 10, atleast 15, or even more S protein variants selected from the groupconsisting of the Q321S, V341I, A348T, N354D, S359N, V367F, K378S,R408I, Q409E, A435S, K458R, I472V, G476S, V483A, Y508H, H519P and D614Gvariants described above. In some embodiments, the panel of different Sprotein variants to which an antibody response is targeted comprises allS protein variants from the group consisting of the Q321S, V341I, A348T,N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, I472V, G476S,V483A, Y508H, H519P and D614G variants described above.

In some embodiments, the panel of different S protein variants to whichan antibody response is targeted comprises at least 5, at least 10, atleast 15, or even more S protein variants selected from the groupconsisting of the Q321L, V341I, A348T, N354D, S359N, V367F, K378R,R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H,H519P and D614G variants described above. In some embodiments, the panelof different S protein variants to which an antibody response istargeted comprises all S protein variants from the group consisting ofthe Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E,A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P and D614Gvariants described above.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises one or more of the mutations described herein for Sprotein variants such as SARS-CoV-2 S protein variants, in particularnaturally occurring S protein variants. In one embodiment, a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein ort the immunogenic variant thereof, e.g., asencoded by the RNA described herein, comprises a mutation at a positioncorresponding to position 501 (N) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises one or more mutations, such as all mutations, of aSARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the groupconsisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. In someembodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, oran immunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof, e.g., as encoded by the RNA described herein, comprisesan amino acid sequence with alanine substitution at position 80, glycinesubstitution at position 215, lysine substitution at position 484,tyrosine substitution at position 501, valine substitution at position701, phenylalanine substitution at position 18, isoleucine substitutionat position 246, asparagine substitution at position 417, glycinesubstitution at position 614, deletions at positions 242 to 244, andproline substitutions at positions 986 and 987 of SEQ ID NO:1. In someembodiments, the methods and agents, e.g., mRNA compositions, describedherein following administration to a subject induce a cell-mediatedimmune response (e.g., CD4⁺ and/or CD8⁺ T cell response). In someembodiments, T cells are induced that recognize one or more eptiopes(e.g., MHC class I-restricted epitopes) selected from the groupconsisting of LPFNDGVYF (SEQ ID NO: 47), GVYFASTEK (SEQ ID NO: 52),YLQPRTFLL (SEQ ID NO: 40), QPTESIVRF (SEQ ID NO: 45), CVADYSVLY (SEQ IDNO: 53), KCYGVSPTK (SEQ ID NO: 54), NYNYLYRLF (SEQ ID NO: 43), FQPTNGVGY(SEQ ID NO: 55), IPFAMQMAY (SEQ ID NO: 46), RLQSLQTYV (SEQ ID NO: 41),GTHWFVTQR (SEQ ID NO: 56), VYDPLQPEL (SEQ ID NO: 57), QYIKWPWYI (SEQ IDNO: 42), and KWPWYIWLGF (SEQ ID NO: 44). In one embodiment, T cells areinduced that recognize the eptiope YLQPRTFLL (SEQ ID NO: 40). In oneembodiment, T cells are induced that recognize the eptiope NYNYLYRLF(SEQ ID NO: 43). In one embodiment, T cells are induced that recognizethe eptiope QYIKWPWYI (SEQ ID NO: 42). In one embodiment, T cells areinduced that recognize the eptiope KCYGVSPTK (SEQ ID NO: 54). In oneembodiment, T cells are induced that recognize the eptiope RLQSLQTYV(SEQ ID NO: 41). In some embodiments, the methods and agents, e.g., mRNAcompositions, described herein are administered according to a regimenwhich achieves such induction of T cells.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration to a subject induce acell-mediated immune response (e.g., CD4⁺ and/or CD8⁺ T cell response)that is detectable 15 weeks or later, 16 weeks or later, 17 weeks orlater, 18 weeks or later, 19 weeks or later, 20 weeks or later, 21 weeksor later, 22 weeks or later, 23 weeks or later, 24 weeks or later or 25weeks or later after administration, e.g., using two doses of the RNAdescribed herein (wherein the second dose may be administered about 21days following administration of the first dose). In some embodiments,the methods and agents, e.g., mRNA compositions, described herein areadministered according to a regimen which achieves such induction of acell-mediated immune response.

In one embodiment, vaccination against Coronavirus described herein,e.g., using RNA described herein which may be administered in theamounts and regimens described herein, e.g., at two doses of 30 μg perdose e.g. administered 21 days apart, may be repeated after a certainperiod of time, e.g., once it is observed that protection againstCoronavirus infection diminishes, using the same or a different vaccineas used for the first vaccination. Such certain period of time may be atleast 6 months, 1 year, two years etc. In one embodiment, the same RNAas used for the first vaccination is used for the second or furthervaccination, however, at a lower dose or a lower frequency ofadministration. For example, the first vaccination may comprisevaccination using a dose of about 30 μg per dose, wherein in oneembodiment, at least two of such doses are administered, (for example, asecond dose may be administered about 21 days following administrationof the first dose) and the second or further vaccination may comprisevaccination using a dose of less than about 30 μg per dose, wherein inone embodiment, only one of such doses is administered. In oneembodiment, a different RNA as used for the first vaccination is usedfor the second or further vaccination, e.g., BNT162b2 is used for thefirst vaccination and BNT162B1 or BNT162b3 is used for the second orfurther vaccination.

In one embodiment, the vaccination regimen comprises a first vaccinationusing at least two doses of the RNA described herein, e.g., two doses ofthe RNA described herein (wherein the second dose may be administeredabout 21 days following administration of the first dose), and a secondvaccination using a single dose or multiple doses, e.g., two doses, ofthe RNA described herein. In various embodiments, the second vaccinationis administered 3 to 24 months, 6 to 18 months, 6 to 12 months, or 5 to7 months after administration of the first vaccination, e.g., after theinitial two-dose regimen. The amount of RNA used in each dose of thesecond vaccination may be equal or different to the amount of RNA usedin each dose of the first vaccination. In one embodiment, the amount ofRNA used in each dose of the second vaccination is equal to the amountof RNA used in each dose of the first vaccination. In one embodiment,the amount of RNA used in each dose of the second vaccination and theamount of RNA used in each dose of the first vaccination is about 30 μgper dose. In one embodiment, the same RNA as used for the firstvaccination is used for the second vaccination. In one embodiment, theRNA used for the first vaccination and for the second vaccination isBNT162b2. In one embodiment, a different RNA as used for the firstvaccination is used for the second vaccination. In one embodiment, theRNA used for the first vaccination is BNT162b2 and the RNA used for thesecond vaccination is RNA encoding a SARS-CoV-2 S protein of aSARS-CoV-2 variant strain, e.g., a strain discussed herein. In oneembodiment, the RNA used for the first vaccination is BNT162b2 and theRNA used for the second vaccination is RNA encoding a SARS-CoV-2 Sprotein of a SARS-CoV-2 variant strain that is prevalent or rapidlyspreading at the time of the second vaccination. In one embodiment, theRNA used for the first vaccination is BNT162b2 and the RNA used for thesecond vaccination is RNA encoding a SARS-CoV-2 S protein, animmunogenic variant thereof, or an immunogenic fragment of theSARS-CoV-2 S protein or the immunogenic variant thereof comprising oneor more of the mutations described herein for S protein variants such asSARS-CoV-2 S protein variants, in particular naturally occurring Sprotein variants. In one embodiment, the RNA used for the firstvaccination is BNT162b2 and the RNA used for the second vaccination isRNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, oran immunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof comprising one or more mutations, such as all mutations,of a SARS-CoV-2 S protein of a SARS-CoV-2 variant selected from thegroup consisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. Inone embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA usedfor the second vaccination is RNA encoding a polypeptide comprising anamino acid sequence with alanine substitution at position 80, glycinesubstitution at position 215, lysine substitution at position 484,tyrosine substitution at position 501, valine substitution at position701, phenylalanine substitution at position 18, isoleucine substitutionat position 246, asparagine substitution at position 417, glycinesubstitution at position 614, deletions at positions 242 to 244, andproline substitutions at positions 986 and 987 of SEQ ID NO:1. In oneembodiment, the vaccination regimen comprises a first vaccination usingtwo doses of RNA encoding a polypeptide comprising an amino acidsequence with proline residue substitutions at positions 986 and 987 ofSEQ ID NO:1 administered about 21 days apart and a second vaccinationusing a single dose or multiple doses of RNA encoding a polypeptidecomprising an amino acid sequence with proline residue substitutions atpositions 986 and 987 of SEQ ID NO:1 administered about 6 to 12 monthsafter administration of the first vaccination, i.e., after the initialtwo-dose regimen. In one embodiment, each RNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationusing two doses of RNA encoding a polypeptide comprising an amino acidsequence with proline residue substitutions at positions 986 and 987 ofSEQ ID NO:1 administered about 21 days apart and a second vaccinationusing a single dose or multiple doses of RNA encoding a polypeptidecomprising an amino acid sequence with alanine substitution at position80, glycine substitution at position 215, lysine substitution atposition 484, tyrosine substitution at position 501, valine substitutionat position 701, phenylalanine substitution at position 18, isoleucinesubstitution at position 246, asparagine substitution at position 417,glycine substitution at position 614, deletions at positions 242 to 244,and proline substitutions at positions 986 and 987 of SEQ ID NO:1administered about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each RNA dose comprises 30 μg RNA.

In one embodiment, the second vaccination results in a boosting of theimmune response.

In one embodiment, the RNA described herein is co-administered withother vaccines. In some embodiments, an RNA composition described hereinis co-administered with an influenza vaccine. In some embodiments, anRNA composition provided herein and other injectable vaccine(s) areadministered at different times. In some embodiments, an RNA compositionprovided herein is administered at the same time as other injectablevaccine(s). In some such embodiments, an RNA composition provided hereinand at least one another injectable vaccine(s) are administered atdifferent injection sites. In some embodiments, an RNA compositionprovided herein is not mixed with any other vaccine in the same syringe.In some embodiments, an RNA composition provided herein is not combinedwith other coronavirus vaccines as part of vaccination againstcoronavirus, e.g., SARS-CoV-2.

The term “disease” refers to an abnormal condition that affects the bodyof an individual. A disease is often construed as a medical conditionassociated with specific symptoms and signs. A disease may be caused byfactors originally from an external source, such as infectious disease,or it may be caused by internal dysfunctions, such as autoimmunediseases. In humans, “disease” is often used more broadly to refer toany condition that causes pain, dysfunction, distress, social problems,or death to the individual afflicted, or similar problems for those incontact with the individual. In this broader sense, it sometimesincludes injuries, disabilities, disorders, syndromes, infections,isolated symptoms, deviant behaviors, and atypical variations ofstructure and function, while in other contexts and for other purposesthese may be considered distinguishable categories. Diseases usuallyaffect individuals not only physically, but also emotionally, ascontracting and living with many diseases can alter one's perspective onlife, and one's personality.

In the present context, the term “treatment”, “treating” or “therapeuticintervention” relates to the management and care of a subject for thepurpose of combating a condition such as a disease or disorder. The termis intended to include the full spectrum of treatments for a givencondition from which the subject is suffering, such as administration ofthe therapeutically effective compound to alleviate the symptoms orcomplications, to delay the progression of the disease, disorder orcondition, to alleviate or relief the symptoms and complications, and/orto cure or eliminate the disease, disorder or condition as well as toprevent the condition, wherein prevention is to be understood as themanagement and care of an individual for the purpose of combating thedisease, condition or disorder and includes the administration of theactive compounds to prevent the onset of the symptoms or complications.

The term “therapeutic treatment” relates to any treatment which improvesthe health status and/or prolongs (increases) the lifespan of anindividual. Said treatment may eliminate the disease in an individual,arrest or slow the development of a disease in an individual, inhibit orslow the development of a disease in an individual, decrease thefrequency or severity of symptoms in an individual, and/or decrease therecurrence in an individual who currently has or who previously has hada disease.

The terms “prophylactic treatment” or “preventive treatment” relate toany treatment that is intended to prevent a disease from occurring in anindividual. The terms “prophylactic treatment” or “preventive treatment”are used herein interchangeably.

The terms “individual” and “subject” are used herein interchangeably.They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog,cat, cattle, swine, sheep, horse or primate) that can be afflicted withor is susceptible to a disease or disorder but may or may not have thedisease or disorder. In many embodiments, the individual is a humanbeing. Unless otherwise stated, the terms “individual” and “subject” donot denote a particular age, and thus encompass adults, elderlies,children, and newborns. In some embodiments, the term “subject” includeshumans of age of at least 50, at least 55, at least 60, at least 65, atleast 70, or older. In some embodiments, the term “subject” includeshumans of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70.In embodiments of the present disclosure, the “individual” or “subject”is a “patient”.

The term “patient” means an individual or subject for treatment, inparticular a diseased individual or subject.

In one embodiment of the disclosure, the aim is to provide an immuneresponse against coronavirus, and to prevent or treat coronavirusinfection.

A pharmaceutical composition comprising RNA encoding a peptide orprotein comprising an epitope may be administered to a subject to elicitan immune response against an antigen comprising said epitope in thesubject which may be therapeutic or partially or fully protective. Aperson skilled in the art will know that one of the principles ofimmunotherapy and vaccination is based on the fact that animmunoprotective reaction to a disease is produced by immunizing asubject with an antigen or an epitope, which is immunologically relevantwith respect to the disease to be treated. Accordingly, pharmaceuticalcompositions described herein are applicable for inducing or enhancingan immune response. Pharmaceutical compositions described herein arethus useful in a prophylactic and/or therapeutic treatment of a diseaseinvolving an antigen or epitope.

As used herein, “immune response” refers to an integrated bodilyresponse to an antigen or a cell expressing an antigen and refers to acellular immune response and/or a humoral immune response. The immunesystem is divided into a more primitive innate immune system, andacquired or adaptive immune system of vertebrates, each of whichcontains humoral and cellular components.

“Cell-mediated immunity”, “cellular immunity”, “cellular immuneresponse”, or similar terms are meant to include a cellular responsedirected to cells characterized by expression of an antigen, inparticular characterized by presentation of an antigen with class I orclass II MHC. The cellular response relates to immune effector cells, inparticular to cells called T cells or T lymphocytes which act as either“helpers” or “killers”. The helper T cells (also termed CD4⁺ T cells)play a central role by regulating the immune response and the killercells (also termed cytotoxic T cells, cytolytic T cells, CD8⁺ T cells orCTLs) kill diseased cells such as virus-infected cells, preventing theproduction of more diseased cells.

An immune effector cell includes any cell which is responsive to vaccineantigen. Such responsiveness includes activation, differentiation,proliferation, survival and/or indication of one or more immune effectorfunctions. The cells include, in particular, cells with lytic potential,in particular lymphoid cells, and are preferably T cells, in particularcytotoxic lymphocytes, preferably selected from cytotoxic T cells,natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.Upon activation, each of these cytotoxic lymphocytes triggers thedestruction of target cells. For example, cytotoxic T cells trigger thedestruction of target cells by either or both of the following means.First, upon activation T cells release cytotoxins such as perforin,granzymes, and granulysin. Perforin and granulysin create pores in thetarget cell, and granzymes enter the cell and trigger a caspase cascadein the cytoplasm that induces apoptosis (programmed cell death) of thecell. Second, apoptosis can be induced via Fas-Fas ligand interactionbetween the T cells and target cells.

The term “effector functions” in the context of the present inventionincludes any functions mediated by components of the immune system thatresult, for example, in the neutralization of a pathogenic agent such asa virus and/or in the killing of diseased cells such as virus-infectedcells. In one embodiment, the effector functions in the context of thepresent invention are T cell mediated effector functions. Such functionscomprise in the case of a helper T cell (CD4⁺ T cell) the release ofcytokines and/or the activation of CD8⁺ lymphocytes (CTLs) and/or Bcells, and in the case of CTL the elimination of cells, i.e., cellscharacterized by expression of an antigen, for example, via apoptosis orperforin-mediated cell lysis, production of cytokines such as IFN-γ andTNF-α, and specific cytolytic killing of antigen expressing targetcells.

The term “immune effector cell” or “immunoreactive cell” in the contextof the present invention relates to a cell which exerts effectorfunctions during an immune reaction. An “immune effector cell” in oneembodiment is capable of binding an antigen such as an antigen presentedin the context of MHC on a cell or expressed on the surface of a celland mediating an immune response. For example, immune effector cellscomprise T cells (cytotoxic T cells, helper T cells, tumor infiltratingT cells), B cells, natural killer cells, neutrophils, macrophages, anddendritic cells. Preferably, in the context of the present invention,“immune effector cells” are T cells, preferably CD4⁺ and/or CD8⁺ Tcells, most preferably CD8⁺ T cells. According to the invention, theterm “immune effector cell” also includes a cell which can mature intoan immune cell (such as T cell, in particular T helper cell, orcytolytic T cell) with suitable stimulation. Immune effector cellscomprise CD34⁺ hematopoietic stem cells, immature and mature T cells andimmature and mature B cells. The differentiation of T cell precursorsinto a cytolytic T cell, when exposed to an antigen, is similar toclonal selection of the immune system.

A “lymphoid cell” is a cell which is capable of producing an immuneresponse such as a cellular immune response, or a precursor cell of suchcell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts,and plasma cells. A lymphoid cell may be an immune effector cell asdescribed herein. A preferred lymphoid cell is a T cell.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs,CD8+ T cells) which comprise cytolytic T cells. The term“antigen-specific T cell” or similar terms relate to a T cell whichrecognizes the antigen to which the T cell is targeted and preferablyexerts effector functions of T cells.

T cells belong to a group of white blood cells known as lymphocytes, andplay a central role in cell-mediated immunity. They can be distinguishedfrom other lymphocyte types, such as B cells and natural killer cells bythe presence of a special receptor on their cell surface called T cellreceptor (TCR). The thymus is the principal organ responsible for thematuration of T cells. Several different subsets of T cells have beendiscovered, each with a distinct function.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T cells and macrophages, among other functions. These cellsare also known as CD4+ T cells because they express the CD4 glycoproteinon their surface. Helper T cells become activated when they arepresented with peptide antigens by MHC class II molecules that areexpressed on the surface of antigen presenting cells (APCs). Onceactivated, they divide rapidly and secrete small proteins calledcytokines that regulate or assist in the active immune response.

Cytotoxic T cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T cells since they express the CD8 glycoprotein on theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body. A majority of T cells have a T cell receptor(TCR) existing as a complex of several proteins. The TCR of a T cell isable to interact with immunogenic peptides (epitopes) bound to majorhistocompatibility complex (MHC) molecules and presented on the surfaceof target cells. Specific binding of the TCR triggers a signal cascadeinside the T cell leading to proliferation and differentiation into amaturated effector T cell. The actual T cell receptor is composed of twoseparate peptide chains, which are produced from the independent T cellreceptor alpha and beta (TCRα and TCRβ) genes and are called α- andβ-TCR chains. γδ T cells (gamma delta T cells) represent a small subsetof T cells that possess a distinct T cell receptor (TCR) on theirsurface. However, in γδ T cells, the TCR is made up of one γ-chain andone δ-chain. This group of T cells is much less common (2% of total Tcells) than the αβ T cells.

“Humoral immunity” or “humoral immune response” is the aspect ofimmunity that is mediated by macromolecules found in extracellularfluids such as secreted antibodies, complement proteins, and certainantimicrobial peptides. It contrasts with cell-mediated immunity. Itsaspects involving antibodies are often called antibody-mediatedimmunity. Humoral immunity refers to antibody production and theaccessory processes that accompany it, including: Th2 activation andcytokine production, germinal center formation and isotype switching,affinity maturation and memory cell generation. It also refers to theeffector functions of antibodies, which include pathogen neutralization,classical complement activation, and opsonin promotion of phagocytosisand pathogen elimination.

In humoral immune response, first the B cells mature in the bone marrowand gain B-cell receptors (BCR's) which are displayed in large number onthe cell surface. These membrane-bound protein complexes have antibodieswhich are specific for antigen detection. Each B cell has a uniqueantibody that binds with an antigen. The mature B cells migrate from thebone marrow to the lymph nodes or other lymphatic organs, where theybegin to encounter pathogens. When a B cell encounters an antigen, theantigen is bound to the receptor and taken inside the B cell byendocytosis. The antigen is processed and presented on the B cell'ssurface again by MHC-II proteins. The B cell waits for a helper T cell(TH) to bind to the complex. This binding will activate the TH cell,which then releases cytokines that induce B cells to divide rapidly,making thousands of identical clones of the B cell. These daughter cellseither become plasma cells or memory cells. The memory B cells remaininactive here; later when these memory B cells encounter the sameantigen due to reinfection, they divide and form plasma cells. On theother hand, the plasma cells produce a large number of antibodies whichare released free into the circulatory system. These antibodies willencounter antigens and bind with them. This will either interfere withthe chemical interaction between host and foreign cells, or they mayform bridges between their antigenic sites hindering their properfunctioning, or their presence will attract macrophages or killer cellsto attack and phagocytose them.

The term “antibody” includes an immunoglobulin comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. Each lightchain is comprised of a light chain variable region (abbreviated hereinas VL) and a light chain constant region. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (CIq) of the classicalcomplement system. An antibody binds, preferably specifically binds withan antigen.

Antibodies expressed by B cells are sometimes referred to as the BCR (Bcell receptor) or antigen receptor. The five members included in thisclass of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primaryantibody that is present in body secretions, such as saliva, tears,breast milk, gastrointestinal secretions and mucus secretions of therespiratory and genitourinary tracts. IgG is the most common circulatingantibody. IgM is the main immunoglobulin produced in the primary immuneresponse in most subjects. It is the most efficient immunoglobulin inagglutination, complement fixation, and other antibody responses, and isimportant in defense against bacteria and viruses. IgD is theimmunoglobulin that has no known antibody function, but may serve as anantigen receptor. IgE is the immunoglobulin that mediates immediatehypersensitivity by causing release of mediators from mast cells andbasophils upon exposure to allergen.

An “antibody heavy chain”, as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations.

An “antibody light chain”, as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations, K and A light chains refer to the twomajor antibody light chain isotypes.

The present disclosure contemplates an immune response that may beprotective, preventive, prophylactic and/or therapeutic. As used herein,“induces [or inducing] an immune response” may indicate that no immuneresponse against a particular antigen was present before induction or itmay indicate that there was a basal level of immune response against aparticular antigen before induction, which was enhanced after induction.Therefore, “induces [or inducing] an immune response” includes “enhances[or enhancing] an immune response”.

The term “immunotherapy” relates to the treatment of a disease orcondition by inducing, or enhancing an immune response. The term“immunotherapy” includes antigen immunization or antigen vaccination.

The terms “immunization” or “vaccination” describe the process ofadministering an antigen to an individual with the purpose of inducingan immune response, for example, for therapeutic or prophylacticreasons.

The term “macrophage” refers to a subgroup of phagocytic cells producedby the differentiation of monocytes. Macrophages which are activated byinflammation, immune cytokines or microbial products nonspecificallyengulf and kill foreign pathogens within the macrophage by hydrolyticand oxidative attack resulting in degradation of the pathogen. Peptidesfrom degraded proteins are displayed on the macrophage cell surfacewhere they can be recognized by T cells, and they can directly interactwith antibodies on the B cell surface, resulting in T and B cellactivation and further stimulation of the immune response. Macrophagesbelong to the class of antigen presenting cells. In one embodiment, themacrophages are splenic macrophages.

The term “dendritic cell” (DC) refers to another subtype of phagocyticcells belonging to the class of antigen presenting cells. In oneembodiment, dendritic cells are derived from hematopoietic bone marrowprogenitor cells. These progenitor cells initially transform intoimmature dendritic cells. These immature cells are characterized by highphagocytic activity and low T cell activation potential. Immaturedendritic cells constantly sample the surrounding environment forpathogens such as viruses and bacteria. Once they have come into contactwith a presentable antigen, they become activated into mature dendriticcells and begin to migrate to the spleen or to the lymph node. Immaturedendritic cells phagocytose pathogens and degrade their proteins intosmall pieces and upon maturation present those fragments at their cellsurface using MHC molecules. Simultaneously, they upregulatecell-surface receptors that act as co-receptors in T cell activationsuch as CD80, CD86, and CD40 greatly enhancing their ability to activateT cells. They also upregulate CCR7, a chemotactic receptor that inducesthe dendritic cell to travel through the blood stream to the spleen orthrough the lymphatic system to a lymph node. Here they act asantigen-presenting cells and activate helper T cells and killer T cellsas well as B cells by presenting them antigens, alongside non-antigenspecific co-stimulatory signals. Thus, dendritic cells can activelyinduce a T cell- or B cell-related immune response. In one embodiment,the dendritic cells are splenic dendritic cells.

The term “antigen presenting cell” (APC) is a cell of a variety of cellscapable of displaying, acquiring, and/or presenting at least one antigenor antigenic fragment on (or at) its cell surface. Antigen-presentingcells can be distinguished in professional antigen presenting cells andnon-professional antigen presenting cells.

The term “professional antigen presenting cells” relates to antigenpresenting cells which constitutively express the MajorHistocompatibility Complex class II (MHC class II) molecules requiredfor interaction with naive T cells. If a T cell interacts with the MHCclass II molecule complex on the membrane of the antigen presentingcell, the antigen presenting cell produces a co-stimulatory moleculeinducing activation of the T cell. Professional antigen presenting cellscomprise dendritic cells and macrophages.

The term “non-professional antigen presenting cells” relates to antigenpresenting cells which do not constitutively express MHC class IImolecules, but upon stimulation by certain cytokines such asinterferon-gamma. Exemplary, non-professional antigen presenting cellsinclude fibroblasts, thymic epithelial cells, thyroid epithelial cells,glial cells, pancreatic beta cells or vascular endothelial cells.

“Antigen processing” refers to the degradation of an antigen intoprocession products, which are fragments of said antigen (e.g., thedegradation of a protein into peptides) and the association of one ormore of these fragments (e.g., via binding) with MHC molecules forpresentation by cells, such as antigen presenting cells to specific Tcells.

The term “disease involving an antigen” refers to any disease whichimplicates an antigen, e.g. a disease which is characterized by thepresence of an antigen. The disease involving an antigen can be aninfectious disease. As mentioned above, the antigen may be adisease-associated antigen, such as a viral antigen. In one embodiment,a disease involving an antigen is a disease involving cells expressingan antigen, preferably on the cell surface.

The term “infectious disease” refers to any disease which can betransmitted from individual to individual or from organism to organism,and is caused by a microbial agent (e.g. common cold). Infectiousdiseases are known in the art and include, for example, a viral disease,a bacterial disease, or a parasitic disease, which diseases are causedby a virus, a bacterium, and a parasite, respectively. In this regard,the infectious disease can be, for example, hepatitis, sexuallytransmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis,HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B,hepatitis C, cholera, severe acute respiratory syndrome (SARS), the birdflu, and influenza.

Certain Exemplary Embodiments

1. A method of immunizing against SARS-CoV-2, the method comprisingsteps of: administering a composition comprising a lipid nanoparticleencapsulated mRNA that encodes at least an epitope of aSARS-CoV-2-encoded polypeptide, according to a regimen established toachieve detectable antibody titer against the epitope in serum within 7days, which regimen comprises administration of at least one dose of thecomposition.2. The method of embodiment 1, wherein the regimen comprisesadministration of at least two doses of the composition.3. The method of embodiment 1, wherein the regimen consists ofadministration of two doses of the composition.4. The method of embodiment 2 or embodiment 3, wherein the first dose isa different amount that one or more subsequent doses.5. The method of embodiment 1 or embodiment 4, wherein the first dose isadministered a period of time before the subsequent dose, which periodof time is at least 1 week, 1 month, 2 months, 3 months, 6 months, 1year, 2 years, 3 years or more.6. The method of any one of embodiments 1-6, wherein the regimen hasbeen established to have an incidence of adverse events below 60% whenadministered to a relevant population of adults.7. The method of embodiment 6, wherein the regimen has been establishednot to elicit local injection site reactions above moderate severitywith an incidence greater than about 1 in 75.8. The method of any one of embodiments 1-7, wherein each dose is nomore than 60 ug or lower, including, e.g., no more than 50 ug, no morethan 40 ug, no more than 30 ug, no more than 20 ug, no more than 10 ug,no more than 5 ug, no more than 2.5 ug, no more than 1 ug.9. The method of any one of embodiments 1-8, wherein each dose is atleast 1 ug or higher, including, e.g., at least 2 ug, at least 5 ug, atleast 10 ug, at least 20 ug, at least 30 ug, at least 40 ug, or more.10. A method comprising administering to a subject a compositioncomprising a lipid nanoparticle encapsulated mRNA, wherein the mRNAencodes an amino acid sequence comprising SARS-COV2 S protein or afragment thereof, wherein the composition is administered in aneffective amount to induce in the subject a SARS-COV-2 S-proteinspecific immune response, wherein the effective amount is sufficient toprovide sterilizing immunity in the subject at an at least 2-fold(including, e.g., at least 3-fold, at least 4-fold, at least 5-fold)lower dose relative to a reference composition (e.g., a reference RNAvaccine or composition).11. A method comprising administering to a subject a compositioncomprising a lipid nanoparticle encapsulated mRNA, wherein the mRNAencodes an amino acid sequence comprising SARS-COV2 S protein or afragment thereof, wherein the composition is administered in aneffective amount to reduce viral load in the subject by at least 80%,relative to a control, at 2 days or more (including, e.g., 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, or more) followingexposure to SARS-COV2, wherein the control is the viral load in asubject administered a reference composition (e.g., a reference RNAvaccine or composition).12. A method comprising administering to a subject a compositioncomprising a lipid nanoparticle encapsulated mRNA, wherein the mRNAencodes an amino acid sequence comprising SARS-COV2 S protein or afragment thereof, wherein the composition is administered in aneffective amount to induce in the subject a SARS-COV-2 S-proteinspecific immune response, wherein efficacy of the RNA vaccine is atleast 80% relative to unvaccinated control subjects.13. The method of any one of embodiments 10-12, wherein the effectiveamount is sufficient to produce detectable levels of SARS-COV-2 Sprotein or a fragment thereof as measured in serum of the subject at1-72 hours post administration.14. The method of any one of embodiments 10-12, wherein the effectiveamount is sufficient to produce a 1,000-10,000 neutralization titerproduced by neutralizing antibody against the SARS-COV-2 S protein asmeasured in serum of the subject at 1-72 hours post administration.15. The method of any one of embodiments 10-14, wherein ananti-SARS-COV-2 S protein antibody titer produced in the subject isincreased by at least 1 log relative to a control, wherein the controlis an anti-SARS-COV-2 S protein antibody titer produced in a subject whohas not been administered a vaccine against SARS-COV-2.16. The method of any one of embodiments 10-15, wherein theanti-SARS-COV-2 S protein antibody titer produced in the subject isincreased at least 2 times relative to a control, wherein the control isan anti-SARS-COV-2 S protein antibody titer produced in a subject whohas not been administered a vaccine against SARS-COV-2.17. The method of any one of embodiments 1-16, wherein the administeringis performed by intramuscular injection.18. An immunogenic composition comprising a lipid nanoparticleencapsulated RNA (e.g., mRNA) that encodes at least an epitope of aSARS-CoV-2-encoded polypeptide, which vaccine composition has beenestablished to achieve detectable antibody titer against the epitope inserum within 7 days after administration to a population of adult humansubjects according to a regimen that includes administration of at leastone dose of the vaccine composition.19. The immunogenic composition of embodiment 18, wherein at least 80%of the uridines in the RNA have a chemical modification.20. The immunogenic composition of embodiment 18 or 19, wherein 100% ofthe uridines in the RNA have a chemical modification.21. The immunogenic composition of any one of embodiments 18-20, whereinthe 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.22. The immunogenic composition of any one of embodiments 18-21, whereinthe lipid nanoparticles in the composition comprise a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid.23. The immunogenic composition of any one of embodiments 18-22, whereinlipid nanoparticles in the composition comprise a molar ratio of about20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and5-25% non-cationic lipid.24. The immunogenic composition of embodiment 22 or 23, wherein thecationic lipid is an ionizable cationic lipid, the non-cationic lipid isa neutral lipid, and the sterol is a cholesterol.25. The immunogenic composition of any one of embodiments 22 or 23,wherein the cationic lipid is selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate.26. The immunogenic composition of any one of embodiments 18-25, whereinthe RNA comprises a 5′ terminal cap and a chemical modification, and theRNA is formulated or is to be formulated as an lipid nanoparticle.27. The immunogenic composition of any one of embodiments 18-26, whereinthe SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof is linked to a signal peptide.28. The immunogenic composition of embodiment 27, wherein the signalpeptide is selected from the group consisting of: a HuIgGk signalpeptide (METPAQLLFLLLLWLPDTTG (SEQ ID NO: 36)); an IgE heavy chainepsilon-1signal peptide (MDWTWILFLVAAATRVHS (SEQ ID NO: 37)); a Japaneseencephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO:38)) and a VSVg protein signal sequence (MKCLLYLAFLFIGVNCA (SEQ ID NO:39)).29. A method for eliciting an immune response in a subject by activatingT cells in vivo, comprising administering to the subject a compositioncomprising a lipid nanoparticle encapsulated modified nucleoside mRNA,wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 Sprotein or a fragment thereof, thereby activating T cells in vivoagainst infection by SARS-COV2 in the subject.30. A method for eliciting an immune response against SARS-COV-2 in asubject, comprising administering to the subject a compositioncomprising a lipid nanoparticle encapsulated modified nucleoside mRNA,wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 Sprotein or a fragment thereof, wherein the composition elicits anincreased production of the SARS-COV2 polypeptide or fragment thereofproduction, as compared to a composition comprising a correspondingunmodified mRNA.31. A method for eliciting an immune response against SARS-COV-2 in asubject, comprising administering to the subject a compositioncomprising a lipid nanoparticle encapsulated modified nucleoside mRNA,wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 Sprotein or a fragment thereof, wherein the composition elicits increasedantibody titers, as compared to a composition comprising a correspondingunmodified mRNA.32. A method for eliciting an immune response against SARS-COV-2 in asubject, comprising administering at least one dose to the subject acomposition comprising a lipid nanoparticle encapsulated modifiednucleoside mRNA, wherein the mRNA encodes an amino acid sequencecomprising SARS-COV2 S protein or a fragment thereof, wherein thecomposition elicits increased antibody titers in the subject at least 7days after the first dose, as compared to the antibody titers againstSARS-COV-2 prior to administration of the composition.33. A method for eliciting an immune response against SARS-COV-2 in asubject, comprising administering at least one dose to the subject acomposition comprising a lipid nanoparticle encapsulated modifiednucleoside mRNA, wherein the mRNA encodes an amino acid sequencecomprising SARS-COV2 S protein or a fragment thereof, wherein thecomposition elicits increased antibody titers in the subject at least 7days after the first dose, as compared to a composition comprising acorresponding unmodified mRNA.34. The method according to any one of embodiments 29-33, wherein themRNA is present at a purity of 90% or greater.35. The method according to any one of embodiments 29-34, wherein thecomposition does not further comprise a unmodified RNA encodingSARS-COV2 S protein or a fragment thereof.36. The method according to any one of embodiments 29-35, wherein thecomposition is administered at least 2 times, the first and secondadministrations being at least 7 days apart.37. The method according to any one of embodiments 29-36, wherein thesubject is at risk for a SARS-CoV-2 infection.38. The method according to any one of embodiments 29-37, wherein thesubject is undergoing treatment for cardiovascular disease.39. The method according to any one of embodiments 29-37, wherein thesubject is undergoing treatment for diabetes.40. The method according to any one of embodiments 29-37, wherein thesubject is undergoing treatment for chronic cardiopulmonary disease.41. The method according to any one of embodiments 29-37, wherein thesubject is undergoing treatment for chronic renal disease.42. The method according to any one of embodiments 29-41, wherein theimmune response is sustained for at least about 30 days.43. The method according to any one of embodiments 29-42, wherein theimmune response is sustained for at least about 60 days.44. The method according to any one of embodiments 29-43, wherein theimmune response is sustained for at least about 180 days.45. The method according to any one of embodiments 29-44, wherein theimmune response comprises virus neutralizing titer.46. The method according to any one of embodiments 29-45, wherein thesubject is at least 18 years of age.47. The method according to any one of embodiments 29-46, wherein thedose comprises 100 ug or less of mRNA.48. The method according to any one of embodiments 29-47, wherein thedose comprises less than 100 ug of mRNA and the composition elicits animmune response that is greater than the immune response elicited by acomposition comprising at least 100 ug of mRNA.49. The method according to any one of embodiments 29-48, wherein thedose comprises about 30 ug of mRNA.50. The method according to any one of embodiments 29-49, wherein theimmune response comprises antibodies against the receptor binding domainof the S protein of SARS-CoV-2.51. The method according to any one of embodiments 29-50, wherein theimmune response comprises RBD-binding IgG.52. The method according to any one of embodiments 29-50, wherein theSARS-CoV-2 S protein or a fragment thereof comprises a receptor bindingdomain.53. A kit comprising a) a composition comprising a lipid nanoparticleencapsulated mRNA; and b) a temperature monitoring system.54. The kit according to embodiment 53, wherein the temperaturemonitoring system comprises a temperature sensor and a display, whereinwhen the temperature monitoring system displays or warns when thetemperature of the composition attains a temperature above about −80° C.55. The kit according to embodiment 53, wherein the temperaturemonitoring system comprises a temperature sensor and a display, whereinwhen the temperature monitoring system displays or warns when thetemperature of the composition attains a temperature above about −60° C.56. A kit comprising a) a composition comprising a lipid nanoparticleencapsulated mRNA; and b) a light sensor.57. The kit according to embodiment 56, wherein the light sensorcomprises a photosensitive element configured to react to exposure tolight, resulting in a change in a material property of thephotosensitive element.58. The method according to any one of embodiments 29-35, wherein thecomposition is administered at least 2 times, the first and secondadministrations being at least 14 days apart.59. The method according to any one of embodiments 29-35, wherein thecomposition is administered at least 2 times, the first and secondadministrations being at least 21 days apart.60. The method according to any one of embodiments 29-48, wherein thedose comprises about 10 ug of mRNA.61. The method according to any one of embodiments 29-35, wherein thecomposition is administered at least 2 times, the first and secondadministrations being at least 28 days apart.62. The method according to any one of embodiments 29-35, wherein themRNA encodes any one of the amino acid sequences SEQ ID NO: 3, SEQ IDNO: 5, and SEQ ID NO: 7.63. An immunogenic composition comprising a messenger ribonucleic acid(mRNA) polynucleotide comprising an open reading frame encoding apolypeptide that comprises a receptor-binding portion of a SARs-CoV-2 Sprotein formulated in at least one lipid nanoparticle comprising acationic lipid, in an effective amount to induce an immune response in asubject administered at least one dose of the immunogenic composition,wherein the isolated mRNA polynucleotide is not self-replicating RNA.64. The immunogenic composition of embodiment 63, wherein the lipidnanoparticle further comprises any one of a non-cationic lipid, sterol,and PEG-modified lipid.65. The immunogenic composition of embodiment 63, comprising an isolatedmessenger ribonucleic acid (mRNA) polynucleotide comprising an openreading frame encoding a polypeptide that comprises a receptor-bindingportion of a SARs-CoV-2 S protein; formulated in at least one lipidnanoparticle that comprises a molar ratio of 20-60% ionizable cationiclipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modifiedlipid, in an effective amount to induce an immune response in a subjectadministered at least one dose of the immunogenic composition, whereinthe isolated mRNA polynucleotide is not self-replicating RNA.66. The immunogenic composition of embodiment 63, wherein thepolypeptide does not comprise the complete S protein.67. The immunogenic composition of embodiment 63, wherein thepolypeptide comprises the receptor binding domain (RBD) of a SARs-CoV-2S protein.68. The immunogenic composition of embodiment 63, wherein thepolypeptide comprises SEQ ID NO: 5.69. The immunogenic composition of embodiment 63, wherein thepolypeptide comprises SEQ ID NO: 29 or 31.70. The immunogenic composition of embodiment 63, wherein thepolypeptide comprises SEQ ID NO: 3.71. The immunogenic composition of embodiment 63, wherein thepolypeptide comprises SEQ ID NO: 7.72. The immunogenic composition of any one of embodiments 63-71, whereinthe isolated mRNA polynucleotide further comprises a 5′ terminal cap,7mG(5′)ppp(5′)NlmpNp.73. The immunogenic composition of any one of embodiments 63-72, whereinat least 80% of the uracil in the open reading frame have a chemicalmodification selected from N1-methyl-pseudouridine orN1-ethyl-pseudouridine.74. The immunogenic composition of any one of embodiments 63-73, whereinthe chemical modification is in the 5-position of the uracil.75. The immunogenic composition of any one of embodiments 63-74, whereinthe efficacy of the immunogenic composition in vaccinated subjects is atleast 60%, relative to unvaccinated subjects, following a single dose ofthe immunogenic composition.76. The immunogenic composition of embodiment 75, wherein the efficacyof the immunogenic composition in vaccinated subjects is at least 70%,relative to unvaccinated subjects, following a single dose of theimmunogenic composition.77. The immunogenic composition of embodiment 75, wherein the efficacyof the immunogenic composition in vaccinated subjects is at least 80%,relative to unvaccinated subjects, following a single dose of theimmunogenic composition.78. The immunogenic composition of embodiment 75, wherein the efficacyof the immunogenic composition in vaccinated subjects is at least 90%,relative to unvaccinated subjects, following a single dose of theimmunogenic composition.79. The immunogenic composition of any one of embodiments 63-78, whereinthe effective amount is sufficient to produce detectable levels of apolypeptide that comprises a receptor-binding portion of a SARS-CoV-2 Sprotein as measured in serum of a subject vaccinated with at least onedose of the immunogenic composition at 1-72 hours post administration.80. The immunogenic composition of any one of embodiments 63-79, whereinthe effective amount is sufficient to produce a 1,000-10,000neutralization titer produced by neutralizing antibody against theantigenic polypeptide that comprises a receptor-binding portion of aSARS-CoV-2 S protein as measured in serum of a subject vaccinated withat least one dose of the immunogenic composition at 1-72 hours postadministration.81. The immunogenic composition of embodiment 80, wherein the1,000-10,000 neutralization titer is produced in the absence ofantibody-dependent enhancement (ADE) of a SARS-CoV-2-associated disease.82. The immunogenic composition of any one of embodiments 63-81, whereinthe effective amount does not induce the immunogeniccomposition-associated enhanced respiratory disease (ERD).83. The immunogenic composition of any one of embodiments 63-82, whereinthe effective amount reduces the amount of SARS-CoV-2 viral RNA in alung of the subject after infection with a SARS-CoV-2 virus, as comparedto the amount of SARS-CoV-2 viral RNA in a lung of an unvaccinatedsubject after infection with a SARS-CoV-2 virus.84. The immunogenic composition of any one of embodiments 63-82, whereinthe effective amount reduces the amount of SARS-CoV-2 viral RNA in alung of the subject at least 3 days after infection with a SARS-CoV-2virus, as compared to the amount of SARS-CoV-2 viral RNA in a lung ofthe subject 3 days after infection with a SARS-CoV-2 virus.85. The immunogenic composition of any one of embodiments 63-82, whereinthe effective amount reduces the amount of SARS-CoV-2 viral RNA in anasal swab sample of the subject after infection with a SARS-CoV-2virus, as compared to the amount of SARS-CoV-2 viral RNA in a nasal swabsample of an unvaccinated subject after infection with a SARS-CoV-2virus.86. The immunogenic composition of any one of embodiments 63-82, whereinthe effective amount does not increase the amount of SARS-CoV-2 viralRNA in a nasal swab sample of the subject 3 days after infection with aSARS-CoV-2 virus, as compared to the amount of SARS-CoV-2 viral RNA in anasal swab sample of the subject 1 day after infection with a SARS-CoV-2virus.87. The immunogenic composition of any one of embodiments 63-87, whereinan anti-SARS-CoV-2 antibody titer produced in a subject vaccinated withat least one dose of the immunogenic composition is increased by atleast 1 log relative to a control, wherein the control is ananti-SARS-CoV-2 antibody titer produced in a subject who has not beenadministered an immunogenic composition against SARS-CoV-2.88. The immunogenic composition of any one of embodiments 63-87, whereinan anti-SARS-CoV-2 antibody titer produced in a subject vaccinated withat least one dose of the immunogenic composition is increased at least 2times relative to a control, wherein the control is an anti-SARS-CoV-2antibody titer produced in a subject who has not been administered animmunogenic composition against SARS-CoV-2.89. The immunogenic composition of any one of embodiments 63-88, whereinthe effective amount is a total dose of 2 μg-100 μg.90. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 100 μg.91. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 20 μg-50 μg.92. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 10 μg-30 μg.93. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 10 μg.94. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 20 μg.95. The immunogenic composition of embodiment 89, wherein the effectiveamount is a total dose of 30 μg.96. The immunogenic composition of any one of embodiments 63-95, whereinthe composition is formulated in a single-dose vial.97. The immunogenic composition of any one of embodiments 63-95, whereinthe composition is formulated in a multi-dose vial.98. The immunogenic composition of any one of embodiments 63-97, whereinintramuscular administration of the effective amount of the immunogeniccomposition to a subject induces a neutralizing antibody titer in asubject.99. The immunogenic composition of embodiment 98, wherein theneutralizing antibody titer is sufficient to reduce viral infection of Bcells by at least 50% relative to a neutralizing antibody titer of anunvaccinated control subject or relative to a neutralizing antibodytiter of a subject vaccinated with a live attenuated viral vaccine, aninactivated viral vaccine, or a protein subunit viral vaccine.100. The immunogenic composition of embodiment 98 or 99, wherein theneutralizing antibody titer is induced in the subject following fewerthan three doses of the immunogenic composition.101. The immunogenic composition of any one of embodiments 98-100,wherein the neutralizing antibody titer and/or a T cell immune responseis sufficient to reduce the rate of asymptomatic viral infectionrelative to the neutralizing antibody titer of unvaccinated controlsubjects.102. The immunogenic composition of any one of embodiments 98-101,wherein the neutralizing antibody titer and/or a T cell immune responseis sufficient to prevent viral latency in the subject.103. The immunogenic composition of any one of embodiments 98-102,wherein the neutralizing antibody titer is sufficient to block fusion ofvirus with epithelial cells and/or B cells of the subject.104. The immunogenic composition of any one of embodiments 63-103,wherein intramuscular administration of the effective amount of theimmunogenic composition to a subject induces a T cell immune response inthe subject.105. The immunogenic composition of embodiment 104, wherein the T cellimmune response comprises a CD4⁺ T cell immune response and/or a CD8⁺ Tcell immune response.106. The immunogenic composition of any one of embodiments 63-105,wherein the encoded polypeptide is presented on the surface of cells ofthe subject.107. A method comprising administering to a subject an immunogeniccomposition of any one of embodiments 63-106, wherein the immunogeniccomposition is administered to the subject in an effective amount toinduce an immune response in the subject.108. The method of embodiment 107, wherein the immune response isinduced against a SARs-CoV-2 virus having a mutation in the RBD, ascompared to SEQ ID NO: 5.109. The method of embodiment 107, wherein the immune response isinduced against a SARs-CoV-2 virus having a mutation in the spikeprotein, as compared to SEQ ID NO: 1.110. The method of embodiment 108 or 109, wherein the immune response isinduced against a SARs-CoV-2 virus having any one of the followingmutations in the RBD: Q321L, V341I, A348T, N354D, S359N, V367F, K378R,R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H,and H519P, as compared to SEQ ID NO: 1.111. The method of embodiment 109, wherein the immune response isinduced against a SARs-CoV-2 virus having a D614G mutation in the spikeprotein, as compared to SEQ ID NO: 1.112. The method of embodiment 107, wherein the immunogenic compositionis administered to the subject annually.113. An RNA, optionally complexed by a (poly)cationic polymer,polyplex(es), protein(s) or peptide(s), which RNA: (a) comprises an openreading frame encoding a polypeptide that comprises areceptor-binding-portion of a SARS-CoV-2 S protein; and (b) is suitablefor intracellular expression of the polypeptide.114. The RNA of embodiment 113, wherein the polypeptide does notcomprise the complete S protein.115. The RNA of embodiment 113 or 114, wherein the RNA further comprisesa 5′ terminal cap, 7mG(5′)ppp(5′)NlmpNp.116. The RNA of any one of embodiments 113-115, wherein at least 80% ofthe uracil in the open reading frame have a chemical modificationselected from N1-methyl-pseudouridine or N1-ethyl-pseudouridine.117. The RNA of any one of embodiments 113-116, wherein the chemicalmodification is in the 5-position of the uracil.118. The RNA of any one of embodiments 113-117 for use in inducing animmune response in humans or vaccinating humans.119. The RNA for use of embodiment 118, wherein the humans comprisehumans known to have been exposed to SARS-CoV-2.120. The RNA for use of embodiment 118, wherein the humans comprisehumans known to have been infected by SARS-CoV-2.121. The RNA for use of embodiment 118, wherein the humans comprisehumans not known to have been exposed to SARS-CoV-2.122. Use of the RNA of any one of embodiments 113-117 for vaccinatinghumans.123. The use of embodiment 122, wherein the humans comprise humans knownto have been exposed to SARS-CoV-2.124. The use of embodiment 122, wherein the humans comprise humans knownto have been infected by SARS-CoV-2.125. The use of embodiment 122, wherein the humans comprise humans notknown to have been exposed to SARS-CoV-2.126. A single-dose formulation comprising the immunogenic composition ofany one of embodiments 63-106.127. A multi-dose formulation comprising the immunogenic composition ofany one of embodiments 63-106 in one vial.128. The formulation according to embodiment 126, comprising at least 2doses per vial.129. The formulation according to embodiment 126, comprising a total of2-12 doses per vial.130. The formulation according to any one of embodiments 126-129,wherein each dose is equal in volume.131. The formulation according to any one of embodiments 126-130,wherein each formulation comprises a total volume of 1-3 mL in the vial.132. The formulation according to any one of embodiments 126-131,wherein the immunogenic composition is frozen.133. A pre-filled vaccine delivery device comprising the immunogeniccomposition of any one of embodiments 63-106.

Citation of documents and studies referenced herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the contents of these documents.

The following description is presented to enable a person of ordinaryskill in the art to make and use the various embodiments. Descriptionsof specific devices, techniques, and applications are provided only asexamples. Various modifications to the examples described herein will bereadily apparent to those of ordinary skill in the art, and the generalprinciples defined herein may be applied to other examples andapplications without departing from the spirit and scope of the variousembodiments. Thus, the various embodiments are not intended to belimited to the examples described herein and shown, but are to beaccorded the scope consistent with the claims.

EXAMPLES Example 1: In Vivo Immunogenicity Using Influenza Hemagglutinin(HA) as a Model Antigen

The potency of the RNA platforms to be used for the coronavirus vaccinedescribed herein has been tested by performing extensive immunogenicityand virus challenge studies using Influenza HA as a model antigen. Thestudies investigated the induction of antibody responses determined withantigen specific enzyme-linked immunosorbent assay (ELISA) test andfunctional studies applying virus-neutralization (VNT) assays. One studyevaluated the potency of the LNP formulation using a modRNA-LNP vaccinethat encodes Influenza HA. Mice were injected IM with 1 μg on days 0 ad28 with an LNP-formulated Influenza HA modRNA. On days 14, 28 and 49blood samples were taken, and tested for immunogenicity. The analysisshowed a high antibody immune response, resulting in very high titers ofantigen-specific IgG in serum and high viral-neutralization activity(FIG. 3 ). Moreover, strong Th1 CD4 and CD8 T cell responses (FIG. 4 )were induced by the modRNA vaccine.

Example 2: Immunogenicity Studies for Coronavirus Vaccine Candidates

Primary pharmacodynamics studies were performed in BALB/c mice to testthe immunogenicity of the vaccine candidates shown in the followingtable.

TABLE 2 Vaccine candidates mRNA Vaccine type Vaccine encoded antigenBNT162a1 uRNA RBD (Receptor Binding Domain) of SARS- CoV-2 Spike protein(S protein) BNT162b1 modRNA RBD (Receptor Binding Domain) of SARS- CoV-2Spike protein (S protein) BNT162b2 modRNA Modified version of SARS-CoV-2Spike protein (S protein) BNT162c1 saRNA RBD (Receptor Binding Domain)of SARS- CoV-2 Spike protein (S protein)

Thus, as can be seen, embodiments of multiple formats were assessed inparallel. This described approach and system achieved remarkable andefficient success, enabling development of an effective clinicalcandidate within several months of provision of antigen (e.g.,SARS-CoV-2 S1 protein and/or RBD thereof) sequence (as described herein,relevant sequence information (e.g., GenBank: MN908947.3) becameavailable in January 2020)

In the study, four groups of each eight female BALB/c mice wereimmunized once with the animal trial material at three different doses,or with buffer (control group; see Table 3). While the clinical trialmaterial will be diluted in saline, the animal trial material wasdiluted in PBS including 300 mM sucrose. As this is the storage bufferof the material itself, the test items are representative for thevaccine that will be used in the planned clinical trials. Immunizationswere given IM using a dose volume of 20 μL.

TABLE 3 Study design Immuni- Dose Blood End of Group No of Vaccinezation volume collection in-life No animals dose Day [μL]/route Dayphase 1 8 buffer 0 20/IM 7, 14, 21 28 2 8 Low 0 20/IM 7, 14, 21 28 3 8Medium 0 20/IM 7, 14, 21 28 4 8 High 0 20/IM 7, 14, 21 28

Blood of immunized animals was collected on days 7, 14, 21 and 28, andanalyzed for the antibody immune response by ELISA and pseudovirus-basedneutralization assay (pVNT).

SARS-CoV-2-S specific antibody responses directed against therecombinant S1 subunit or the RBD were detected by ELISA. In brief, highprotein-binding 96-well plates (MaxiSorp ELISA plates, VWR InternationalGmbH, Cat. No. 7341284) were coated with 100 ng recombinant S1 subunit(Sino Biological Inc., Cat. No. 40591-V08H) or RBD (Sino BiologicalInc., Cat. No. 40592-V02H) per well in 100 μL coating buffer (50 mMsodium carbonate-bicarbonate buffer, pH9.6) overnight at 4° C. Plateswere washed three times with 300 μL/well 1× phosphate-buffered saline(PBS, VWR International GmbH, Cat. No. 0780-10L) supplemented with 0.01%Tween 20 (Carl Roth GmbH & Co. KG, Cat. No. 9127.1) and blocked with 250μL/well 1× Casein Blocking Buffer (Sigma-Aldrich GmbH, Cat No. B6429-500ml) for 1 hour at 37° C. on a microplate shaker. Plates were againwashed three times with 300 μL/well 1×PBS supplemented with 0.01% Tween20 and incubated with mouse serum samples diluted in 1× Casein BlockingBuffer for 1 hour at 37° C. on a microplate shaker. Plates were washedthree times with 300 μL/well 1×PBS supplemented with 0.01% Tween 20 andsubsequently incubated with Peroxidase-conjugated goat anti-mousesecondary antibody (Jackson ImmunoResearch Ltd., Cat. No. 115-036-071;diluted 1:7500 in 1× Casein Blocking Buffer) for 45 minutes at 37° C. ona microplate shaker. Plates were washed three times with 300 μL/well1×PBS supplemented with 0.01% Tween 20 and 100 μL/well TMB substrate(Biotrend Chemiekalien GmbH, Cat. No. 4380A) was added. Plates wereincubated for 8 min at room temperature and the reaction stopped byaddition of 100 μL 25% sulphuric acid (VWR International GmbH, Cat. No.1007161000). Plates were read on a microplate reader and the recordedabsorbance at 450 nm corrected by subtracting the reference absorbanceat 620 nM.

Functional antibody responses to the vaccine candidates were detected bypVNT. The pVNT uses a replication-deficient vesicular stomatitis virus(VSV) that lacks the genetic information for the VSV envelopeglycoprotein G but contains an open-reading frame (ORF) for greenfluorescent protein (GFP). VSV/SARS-CoV-2 pseudovirus was generatedaccording to a published protocol (Hoffmann et al., Cell, 2020; PMID32142651). The pseudotype virus bears the SARS-CoV-2 S protein, whichmediates cell entry. Therefore, the pseudovirus can be inactivated byneutralizing antibodies that bind SARS-CoV-2 S. This inactivation can beanalyzed via in vitro methods.

In brief, 4×10⁴ Vero 76 cells (ATCC® CRL-1587™) per well were seeded ina 96-well plate (Greiner Bio-One GmbH, Cat. No. 655160) in 150 μL/wellDMEM (Thermo Fisher Scientific, Cat. No. 61965059) supplemented with 10%fetal bovine serum (FBS, Sigma-Aldrich GmbH, Cat. No. F7524). Cells wereincubated for 4 to 6 hours at 37° C. and 7.5% CO₂. Meanwhile, mouseserum samples were diluted 1:6 up to 1:768 in DMEM/10% FBS in two-folddilution steps. Diluted serum samples were combined with an equal volumeof titrated and pre-diluted VSV/SARS CoV-2 pseudovirus supernatant,resulting in a serum dilution ranging from 1:12 up to 1:1536. Thepseudovirus/serum dilution mix was incubated for 5 min at RT on amicroplate shaker at 750 rpm with an additional 5 min incubation at RTwithout agitation. 50 μL/well pseudovirus/serum dilution mix was addedto the seeded Vero-76 cells with the applied pseudovirus volume per wellcorresponding to 200 infectious units (IU). Each dilution of serumsamples was tested in duplicate wells. Cells were incubated for 16 to 24hours at 37° C. and 7.5% CO₂. Vero 76 cells incubated with pseudovirusin the absence of mouse sera were used as positive controls. Vero 76cells incubated without pseudovirus were used as negative controls.After the incubation, the cell culture plates were removed from theincubator, placed in an IncuCyte Live Cell Analysis system (EssenBioscience) and incubated for 30 min prior to the analysis. Whole wellscanning for brightfield and GFP fluorescence was performed using a 4×objective. To calculate the neutralizing titer, infected GFP-positivecell number per well was compared with the pseudovirus positive control.Mean values of the pseudovirus positive control multiplied by 0.5represent the pseudovirus neutralization 50% (pVN50). Serum samples withmean values below this cut-off exhibit >50% virus neutralizationactivity, respectively.

Immunogenicity Study of BNT162a1 (RBL063.3)

To dissect the potency of the LNP-formulated uRNA vaccine coding forBNT162a1, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine will be investigated by focusing onthe antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization show anearly, dose-dependent immune activation against the S1 protein and thereceptor binding domain (FIG. 5 ).

Immunogenicity Study of BNT162b1 (RBP020.3)

To dissect the potency of the LNP-formulated modRNA vaccine coding forBNT162b1, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine will be investigated by focusing onthe antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization show anearly, dose-dependent immune activation against the S1 protein and thereceptor binding domain (FIG. 6 ). Sera obtained 14, 21, and 28 d afterimmunization show high SARS-CoV-2 pseudovirus neutralization, especiallysera from mice immunized with 1 or 5 μg BNT162b1 and correlating withthe strong increase of IgG antibody titers (FIG. 7 ).

Immunogenicity Study of BNT162c1 (RBS004.3)

To dissect the potency of the LNP-formulated saRNA vaccine coding forBNT162c1, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine will be investigated by focusing onthe antibody immune response.

ELISA data 7, 14 and 21 d after the first immunization show an early,dose-dependent immune activation against the S1 protein and the receptorbinding domain (FIG. 8 ). Sera obtained 14, and 21 d after immunizationshow dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG.9 ).

Immunogenicity Study of LNP-Formulated uRNA Encoding the Viral SProtein-V8 (SEQ ID NO: 7, 8) (RBL063.1)

To dissect the potency of the LNP-formulated uRNA vaccine coding for theviral S protein-V8 (RBL063.1), BALB/c mice were immunized IM once asoutlined in Table 3. The immunogenicity of the RNA vaccine will beinvestigated by focusing on the antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization are availablethat show an early, dose-dependent immune activation against the S1protein and the receptor binding domain (FIG. 10 ). Sera obtained 14, 21and 28 d after immunization show dose-dependent SARS-CoV-2 pseudovirusneutralization activity (FIG. 11 ).

Immunogenicity Study of BNT162b2 (RBP020.1)

To dissect the potency of the vaccine BNT162b2 (RBP020.1), theimmunogenicity of the construct was investigated. For this purpose, adose titration study in BALB/c mice was initiated where the immuneresponse will be analyzed focusing on the antibody immune response.

ELISA data 7, 14, and 21 d after the first immunization are availablethat show an early, dose-dependent immune activation against the S1protein and the receptor binding domain (FIG. 12 ). Sera obtained 14,and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirusneutralization activity (FIG. 13 ).

Immunogenicity Study of the LNP-Formulated saRNA Encoding the Viral SProtein-V9 (SEQ ID NO: 7, 9) (RBS004.2)

To dissect the potency of the LNP-formulated saRNA vaccine coding forV9, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine will be investigated by focusing onthe antibody immune response.

ELISA data 7, 14, and 21 d after the first immunization are availablethat show an early, dose-dependent immune activation against the S1protein and the receptor binding domain (FIG. 14 ). Sera obtained 14,and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirusneutralization activity (FIG. 15 ).

The above data demonstrate an immune response for both the RBD with atrimerization domain (“V5”) and the mutated full-length S protein(“V8”/“V9”) in vivo in all tested platforms (including the vaccinesBNT162a1, BNT162b1, BNT162b2, and BNT162c1). The antibody immuneresponse was already seen at very early time points by ELISA (i.e., at 7d post-immunization) Importantly, induced antibodies were able toefficiently neutralize SARS-COV-2 pseudovirus infection in vitro. Also,the induction of an antibody response using a very low immunization doseof 0.2 μg/mouse when using the modRNA platform (BNT162b1, BNT162b2) aswell as the saRNA platform (BNT162c1) indicates a high potency of thevaccine candidates.

In mice, BNT162b2 induced a higher antigen-specific titer compared toBNT162b1 encoded with the identical RNA platform. As expected, theimmunogenicity in mice against the antigens differs between the RNAplatforms. In mice, the most immunogenic platform based onantigen-specific antibody induction is the modRNA followed by saRNA. TheuRNA platform induces the lowest antigen-specific antibody titer.

Example 3: Selection of Formulation

The LNP delivery system was in general developed to effectively andsafely deliver therapeutic nucleic acids into the cytosol of variouscell types after local administration in vivo. The early formulationwork was performed with several promising LNP formulations and surrogateRNA coding for luciferase. The aim of the experiments was to correlatethe effect of different ionizable cationic lipids on the efficacy of RNAdelivery by LNPs in vivo. Formulations were compared in terms of RNAencapsulation efficiency, apparent pKa, LNP size and polydispersity.Among the screened cationic lipids, ALC-0315 exhibited suitable physicalcharacteristics regarding particle size, homogeneity, and RNAencapsulation efficiency.

Based on this the ALC-0315/DSPC/CHOL/ALC-0159 prototype was submittedfor in vivo screening. The results presented in FIG. 16 summarize the invivo testing of two independent pilot batches using luciferase (Luc)RNA. The results demonstrate improved potency of the ALC-0315 prototypeas compared to an internal benchmark (ALC-0218). On the basis of thesestudies, ALC-0315 was identified as a highly potent cationic lipid andbrought forward for further product development studies.

The formulation screening procedure described above involves intravenousadministration resulting in delivery primarily to the liver. Themechanism of LNP uptake into hepatocytes is driven by binding ofendogenous apolipoproteins to the LNP followed by receptor-mediatedendocytosis e.g. through low density lipoprotein receptors. In order toinvestigate whether the same mechanism is involved for an intramuscularadministration, Luc RNA containing LNPs comprising ALC-0315 wereinjected intravenously (0.3 mg/kg) and intramuscularly (0.2 mg/kg) intoApoE knockout mice in the presence or absence of recombinant humanApoE3. As control, wild-type C57Bl/6 mice were also treated by thedifferent routes of administration. RNA-LNP were pre-incubated withrecombinant human ApoE3 (1 mg encapsulated mRNA with 1 mg ApoE3) for 1hour at room temperature (RT) prior to administration. Luc expressionwas monitored at 4, 24, 72 and 96 hours post administration (FIG. 17 ).

When mice were administered intravenously, Luc expression was detectedin the wild-type C57Bl/6 mice. In the ApoE knockout mice Luc expressionwas significantly reduced however when preincubated with exogenous ApoEthe expression of Luc was recovered to similar expression levels aswild-type mice (FIG. 18 ).

In vivo Luc expression experiments using mouse models showed, thatsimilar mechanisms are involved in the uptake of RNA-LNP in case ofintramuscular administration as for intravenous administration, and thisis not only true for hepatocytes but also for the cells local to theadministration site.

In vivo experiments after intramuscular administration of the finalALC-0315/DSPC/CHOL/ALC-0159, confirmed minimal drainage with regards tobiodistribution, immunogenicity (vaccine activity) and tolerability.

Example 4: Immunogenicity Studies for Coronavirus Vaccine Candidates

Functional cellular immune responses to the vaccine candidates weredetected by ELISpot assay using the IFN-γ ELISpot^(PLUS) kit (Mabtech,Cat. No. 3321-4APT-2). In brief, spleens were removed from animals aftersacrifice at day 28 after vaccination. Spleens were mechanicallydissociated using the plunger of a syringe and a 70 μM cell strainer(Greiner Bio-One GmbH, Cat. No. 542070). Splenocytes were washed with anexcess volume of DPBS (Thermo Fisher Scientific, Cat. No. 14190-094)followed by centrifugation at 300×g for 6 min at RT and discarding thesupernatants. Erythrocytes were then lysed with erythrocyte lysis buffer(154 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) for 5 min at RT. The reactionwas stopped with an excess volume of DPBS. After another washing step,cells were resuspended in RPMI 1640 medium (Gibco, Cat. no. 61870-010)supplemented with 10% FBS, 1% MEM Non-Essential Amino Acids Solution(Gibco, Cat. No. 11140-035), 1% sodium pyruvate (Gibco, Cat. No.11360-039), 0.5% penicillin/streptomycin (Gibco, Cat. No. 15140-122),passed through a 70 μm cell mesh again and counted. CD8+ or CD4+ T cellswere isolated from splenocyte cell suspensions using CD8a or CD4 MACS®MicroBeads (Miltenyi Biotec, Cat. No. 130-117-044 and 130-117-043)according to the manufacturer's instructions. In parallel, 96-wellELISpot plates were washed with PBS and blocked with medium (RPMI 1640medium supplemented with 10% FBS, 1% MEM Non-Essential Amino AcidsSolution, 1% sodium pyruvate, 0.5% penicillin/streptomycinmedium) for atleast 30 min at 37° C. 1×10s CD8+ or CD4+ T cells in 100 μL medium weresubsequently re-stimulated by addition of 50 μL peptide solution(irrelevant control peptide AH1 (2 μg/mL; sequence: SPSYVYHQF (SEQ IDNO: 35)), PepMix™ SARS-CoV-2 S-RBD (0.025 μg/mL per peptide; JPT,customized) or PepMix™ SARS-CoV-2 Spike Glycoprotein (0.1 μg/mL perpeptide; JPT, Cat. No. PM-WCPV-S-2) and 50 μL of autologous bonemarrow-derived dendritic cells in the IFN-γ ELISpot assay. Eachcondition was tested in duplicate. Plates were incubated overnight in a37° C. humidified incubator with 5% CO₂ and after approximately 18 h,cells were removed from the plates. IFN-γ spots were detected accordingto the manufacturer's protocol. After plate drying for 2-3 h under thelaminar flow, an ELISpot plate reader (ImmunoSpot® S6 Core Analyzer,CTL) was used to count and analyze spot numbers per well.

In addition to ELISpot assay, Luminex analyses were conducted to informabout the T_(H)1 or T_(H)2 nature of the detected T cell response. 5×10⁵splenocytes in 100 μL RPMI 1640 medium supplemented with 10% FBS, 1% MEMNon-Essential Amino Acids Solution, 1% sodium pyruvate, 0.5%penicillin/streptomycinmedium were transferred to a 96-well flat bottomcell culture plate. 100 μL irrelevant control peptide AH1 (2 μg/mL;sequence: SPSYVYHQF (SEQ ID NO: 35)), or PepMix™ SARS-CoV-2 SpikeGlycoprotein (0.1 μg/mL per peptide; JPT, Cat. No. PM-WCPV-S-2) wereadded. The plates were incubated for 48 hours and supernatant thereafterwas harvested for cytokine profiling. Cytokine concentrations insupernatants of the re-stimulated splenocytes were determined using abead-based T_(H)1/T_(H)2 ProcartaPlex immunoassay (Thermo FisherScientific, Cat. No. EPX110-20820-901) according to the manufacturer'sinstructions. Fluorescence was measured with the Bioplex200 System(Biorad) and analyzed with ProcartaPlex Analyst 1.0 software (ThermoFisher Scientific). The following analytes were measured: IFN-γ;IL-12p70; IL-13; IL-1 beta; IL-2; IL-4; IL-5; IL-6; TNF alpha; GM-CSF;IL-18.

For immunophenotyping, flow cytometry analysis was performed. Briefly,erythrocytes from 50 μL freshly drawn blood were lysed with ACK lysingbuffer (Gibco) and cells were stained with fixable viability dye(eBioscience) and anti-CXCR5 (rat IgG2a) antibody in the presence of Fcblock (both BD Bioscience) in flow buffer (DPBS (Gibco) supplementedwith 2% FCS, 2 mM EDTA (both Sigma) and 0.01% sodium azide (Morphisto)for 20 minutes at room temperature. After staining with anti-rat IgG2abiotin in flow buffer for 20 minutes at 2-8° C., cells were stainedextracellularly with antibodies against CD3, CD4, CD8a, CD38, CD44,PD-1, ICOS, CD62L, CXCR5, CD19 and streptavidin in Brilliant StainBuffer Plus (BD Bioscience) diluted in flow buffer for 20 minutes at2-8° C. Cells were fixed with 2% RotiHistofix (Roth) for 15 minutes atroom temperature. Cells were resuspended in Perm buffer(FoxP3/Transcription Factor Staining Buffer Set, eBioscience) andincubated over night at 2-8° C. Permeabilized cells were intracellularlytreated with Fc block for 10 minutes at 2-8° C. and stained with T-betand GATA (BD Bioscience) antibodies for 30 minutes at 2-8° C. Cells wereresuspended in flow buffer and acquired on a BD Symphony A3 flowcytometer (BD Bioscience) and analyzed with FlowJo 10.6.2.

For mouse B cell subtyping in draining lymph nodes, 2.5×10⁵ lymph nodecells were treated with Fc block for 15 minutes and stainedextracellularly with antibodies against CD19, CD45R/B220, IgD, CD138,IgM, CD38, CD95/FAS, IgG1, IgG2a, CD73, GR-1, F4/80, CD4, CD8 inBrilliant Stain Buffer (BD Bioscience) for 20 minutes at 2-8° C. Cellswere fixed with 2% RotiHistofix and incubated over night at 2-8° C.

Immunogenicity Study of BNT162b1 (RBP020.3)

To dissect the potency of the LNP-formulated modRNA vaccine coding forBNT162b1, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine was investigated by focusing on thecellular immune response.

After stimulation with an S protein- or RBD-specific peptide pool, butnot after stimulation with irrelevant peptide AH1, both CD4+ and CD8+ Tcells displayed IFN-γ responses in the IFN-γ ELISpot assay (FIG. 22 ).In Luminex analysis, cytokine production after peptide stimulation wasconfirmed for analytes that indicate a T_(H)1-driven immune response(FIG. 23 ).

Immunophenotyping analysis of blood 7 days after immunization (FIG. 24 )revealed a significant increase in circulating T follicular helper cells(Tfh) and activated T cells. At day 12 after immunization, draininglymph nodes from immunized BALB/c mice were dissected and B cellsubpopulation analysis was performed (FIG. 25 ). A significant increasein B cells was found in lymph nodes with detectable numbers of plasmacells, class switched B cells and IgG1 or IgG2a positive germinal centerB cells. Both in blood and draining lymph node, an activation andmaturation of the adaptive immune response was confirmed.

Immunogenicity Study of the LNP-Formulated modRNA Encoding the ViralP2-S Protein V8 (RBP020.1)

To dissect the potency of the LNP-formulated modRNA vaccine coding forRBP020.1, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine was investigated by focusing on thecellular immune response.

After stimulation with an S protein-specific peptide pool, but not afterstimulation with irrelevant peptide AH1, both CD4⁺ and CD8⁺ T cellsdisplayed IFN-γ responses in the IFN-γ ELISpot assay (FIG. 26 ). InLuminex analysis, cytokine production after peptide stimulation wasconfirmed for analytes that indicate a T_(H)1-driven immune response(FIG. 27 ).

Immunogenicity Study of the LNP-Formulated saRNA Encoding the Viral P2-SProtein V9 (RBS004.2)

To dissect the potency of the LNP-formulated saRNA vaccine coding forRBS004.2, BALB/c mice were immunized IM once as outlined in Table 3. Theimmunogenicity of the RNA vaccine was investigated by focusing on thecellular immune response.

After stimulation with an S protein-specific peptide pool, but not afterstimulation with irrelevant peptide AH1, both CD4⁺ and CD8⁺ T cellsdisplayed IFN-γ responses in the IFN-γ ELISpot assay (FIG. 28 ). InLuminex analysis, cytokine production after peptide stimulation wasconfirmed for analytes that indicate a T_(H)1-driven immune response(FIG. 29 ).

Immunogenicity Study of BNT162b3 Variants BNT162b3c and BNT162b3d

To get an idea about the potential potency of transmembrane-anchoredRBD-based vaccine antigens (Schematic in FIG. 30 ; BNT162b3c (1) andBNT162b3d (2)), BALB/c mice were immunized IM once with 4 μg LNP-C12formulated mRNA or with buffer as control. The non-clinical LNP-C12formulated mRNAs were used as surrogate for the BNT162b3 variantsBNT162b3c and BNT162b3d. The immunogenicity of the RNA vaccine wasinvestigated by focusing on the antibody immune response.

ELISA data 6, 14 and 21 d after the first immunization show an early,dose-dependent immune activation against the S1 protein and the receptorbinding domain (FIG. 31 ). Sera obtained 6, 14 and 21 d afterimmunization show high SARS-CoV-2 pseudovirus neutralization,correlating with the increase of IgG antibody titers (FIG. 32 ).

Example 5: Immunogenicity Studies for Coronavirus Vaccine Candidates inNon-Human Primates (NHP)

Six Rhesus macaques per group were immunized IM with 30 or 100 μg ofBNT162b1 or with buffer on days 0 and 21. By 14 days after the firstdose, antibodies that bound a recombinant S1 were readily detectable,and levels of S1-binding antibodies exceeded the upper limit ofquantification of the assay (10,000 U/mL) by day 28. For comparison,S1-binding antibodies of 62 human COVID-19 convalescent sera, obtainedafter the onset of symptoms were analyzed. All timepoints throughout thetwo NHP groups significantly exceeded the mean of the human COVID-19convalescent sera of 422 U/mL (FIG. 33 a ). The VNT geometric meantiters (GMTs) of sera from Rhesus macaques immunized with either doselevel of BNT162b1 were detectable by 14 days after a single immunizationand reached a geometric mean of 768 (30 μg dose level) or 1,714 (100 μg)by day 28 (FIG. 33 b ). The neutralization GMTs were 282 (30 μg) and 975(100 μg) on day 35 (14 days after the boost) (FIG. 33 b ). Flowcytometry analysis of CD4⁺ T cells from blood samples stimulated with aS peptide mix on day 42 revealed significant secretion of the T_(H)1cytokines IFNγ, IL-2 and TNFα. Additionally, IL-21 secretion wassignificantly increased. IL-21 is known to play a critical role in Bcell activation, expansion and plasma cell generation as well as thegeneration of Tfh. In contrast, no significant amounts of the T_(H)2cytokine IL-4 were detected (FIG. 33 c ). In summary and in alignment tothe results obtained in mice, BNT162b1 induced a high antibody immunewith an early affinity maturation coming with a T_(H)1 biased immuneresponse.

In summary, we demonstrate that the methyl-nucleoside m1W-modified mRNAencoding the trimeric receptor binding domain from the SARS-CoV-2 Sprotein is protective in non-human primates.

Example 6: Phase 1/2 Study to Describe the Safety, Tolerability, andImmunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1) in HealthyAdults 18 to 55 Years of Age

We report safety, tolerability, and immunogenicity from aplacebo-controlled, observer-blinded dose escalation trial among healthyadults randomized to receive 2 doses of 10 μg, 30 μg, or 100 μg ofBNT162b1, a lipid nanoparticle (LNP)-formulated, nucleoside-modified,mRNA vaccine candidate that encodes trimerized SARS-CoV-2 spikeglycoprotein RBD antigen. Local reactions and systemic events weredose-dependent, generally mild to moderate, and transient. RBD-bindingIgG concentrations and SARS-CoV-2 neutralizing titers increased withdose level and after a second dose. Geometric mean neutralizing titersreached 1.8- to 2.8-fold that of a COVID-19 convalescent human serumpanel.

The BNT162b1 vaccine candidate now being tested clinically incorporatesnucleoside 1-methyl-pseudouridine modified RNA (modRNA) and encodes thereceptor binding domain of the SARS-CoV-2 spike, a key target ofneutralizing antibodies. The RBD antigen expressed by BNT162b1 ismodified by the addition of a T4 fibritin-derived “foldon” trimerizationdomain to increase its immunogenicity. This RNA vaccine candidate isbeing tested in parallel in coordinated studies in Germany and in theUS. Here, we present data obtained in the US study.

Methods

Study Design and Participants:

This Phase 1/2, randomized, placebo-controlled, observer-blinded trialwas conducted in the United States to assess the safety, tolerability,and immunogenicity of ascending dose levels of various BNT162 mRNAvaccine candidates. Assessment of three dose levels (10, 30, or 100 μg)of the BNT162b1 candidate was conducted at two sites in the UnitedStates. This study utilized a sentinel cohort design with progressionand dose escalation taking place after review of data from the sentinelcohort at each dose level. Healthy men and nonpregnant women 18 to 55years of age were enrolled. Key exclusion criteria included individualswith known infection with human immunodeficiency virus, hepatitis Cvirus, or hepatitis B virus; immunocompromised individuals and thosewith a history of autoimmune disease; those with increased risk forsevere COVID-19; previous clinical or microbiological diagnosis ofCOVID-19; receipt of medications intended to prevent COVID-19; previousvaccination with any coronavirus vaccine; and a SARS-CoV-2 NAAT-positivenasal swab within 24 hours before study vaccination.

The final protocol and informed consent document were approved byinstitutional review boards for each of the investigational centersparticipating in this study, and this study was conducted in compliancewith all International Council for Harmonisation (ICH) Good ClinicalPractice (GCP) guidelines and the ethical principles of the Declarationof Helsinki. A signed and dated informed consent was required before anystudy-specific activity was performed.

Endpoints:

The study's primary endpoints included: the proportion of participantsreporting prompted local reactions, systemic events, and use ofantipyretic and/or pain medication within 7 days after vaccination, AEsthrough 1 month after the last dose, and SAEs through 6 months aftervaccination, and the proportion of sentinel cohort participants withclinical laboratory abnormalities 1 week after vaccination and gradingshifts in laboratory assessments between baseline and 1 and 7 days afterDose 1 and between Dose 2 and 7 days after Dose 2. Secondary endpointsincluded: SARS-CoV-2 neutralizing geometric mean titers (GMTs);SARS-CoV-2 S1-binding IgG and RBD-binding IgG geometric meanconcentrations (GMCs) 7 and 21 days after Dose 1 and 7 and 14 days afterDose 2; geometric mean fold rise (GMFR), ≥4-fold rise from baseline andgeometric mean ratio (GMR) of SARS-CoV-2 serum neutralizing GMTs toSARS-CoV-2-antigen binding antibody GMCs at each time point.

Procedures:

Study participants were randomly assigned to a vaccine group using aninteractive web-based response technology system with each groupcomprising 15 participants (12 active vaccine recipients and 3 placeborecipients). Participants received two 0.5-mL doses of either BNT162b1or placebo, administered by intramuscular injection into the deltoidmuscle.

BNT162b1 incorporates a Good Manufacturing Process (GMP)-grade mRNA drugsubstance that encodes the trimerized SARS-CoV-2 spike glycoprotein RBDantigen. The mRNA is formulated with lipids as the mRNA-LNP drugproduct. The vaccine was supplied as a buffered-liquid solution for IMinjection and was stored at −80° C. The placebo was a sterile salinesolution for injection (0.9% sodium chloride injection, in a 0.5-mLdose).

Safety assessments for all participants included 4-hour observationafter vaccination (for the first 5 participants vaccinated in eachsentinel group, and a 30-minute observation (for the remainder ofparticipants) for immediate AEs. The safety assessments also includedself-reporting of prompted local reactions (redness, swelling, and painat the injection site), systemic events (fever, fatigue, headache,chills, vomiting, diarrhea, muscle pain, and joint pain), and the use ofantipyretic and/or pain medication in an electronic diary (e-diary) for7 days after vaccination, reporting of unprompted AEs through 1 monthafter vaccination and SAEs through 6 months after the last vaccination.Hematology and chemistry assessments were conducted at screening, 1 and7 days after Dose 1, and 7 days after Dose 2.

The protocol specified safety stopping rules for all sentinel-cohortparticipants. Both an internal review committee (IRC) and an externaldata monitoring committee (EDMC) reviewed all safety data.

Immunogenicity Testing:

50 mL of blood was collected for immunogenicity assessments (SARS-CoV-2serum neutralization assay, SARS-CoV-2 S1-specific IgG direct Lumineximmunoassay, SARS-CoV-2 RBD-specific IgG direct Luminex immunoassay andnonvaccine antigen (NVA) Ig direct Luminex immunoassay) before eachstudy vaccination, at 7 and 21 days after Dose 1 and at 7, 14, 1 monthand 6 months after Dose 2.

The SARS-CoV-2 neutralization assay used a previously described strainof SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse geneticsand engineered by the insertion of an mNeonGreen (mNG) gene into openreading frame 7 of the viral genome. This reporter virus generatessimilar plaque morphologies and indistinguishable growth curves fromwild-type virus. Serial dilutions of heat inactivated sera wereincubated with the reporter virus for 1 hour at 37° C. beforeinoculating Vero E6 cell monolayers. Infected foci were detected byfluorescence between 16-24 hours after inoculation by the addition ofHoechst 33342 Solution and counted with a Cytation 7 Cell ImagingMulti-Mode Reader.

Statistical Analysis:

The sample size for the sentinel cohort of the study was not based onstatistical hypothesis testing. The primary safety objective wasevaluated by descriptive summary statistics for local reactions,systemic events, abnormal hematology and chemistry laboratoryparameters, and AEs and SAEs for each vaccine group. A 3-tier approachwas used to summarize AEs. The secondary immunogenicity objectives weredescriptively summarized at the various time points.

Results

Between 4 May 2020 and 19 Jun. 2020, 76 subjects were screened, and 45participants were randomized and vaccinated. Twelve participants perdose level (10 μg, 30 μg, or 100 μg) were vaccinated with BNT162b1 onDays 0 and 21, and 9 participants received placebo (FIG. 34 ). The studypopulation consisted of healthy male and nonpregnant femaleparticipants, 18 to 55 years of age with a mean age of 35.4 years(minimum 19 and maximum 54 years). Overall, 51.1% of participants weremale and 48.9% were female. Most participants were white (82.2%) andnon-Hispanic/non-Latino (93.3%).

Safety and Tolerability

In the 7 days after either vaccination, pain at the injection site wasthe most frequently local reaction, reported by 58.3% (7/12) in the 10μg and 100.0% (12/12 each) in the 30 μg and 100 μg BNT162b1 groups andby 22.2% (2/9) of placebo recipients after Dose 1 and by 83.3% and100.0% of BNT162b1 recipients at the 10 μg and 30 μg dose levels,respectively, after Dose 2. All local reactions were mild or moderate inseverity except for one report of severe pain following Dose 1 of 100 μgBNT162b1.

The most common systemic events reported in the 7 days after vaccinationwere mild to moderate fatigue and headache in both BNT162b1 and placeborecipients. Systemic events increased with dose level and were reportedin a greater number of subjects after the second dose (10 μg and 30 μggroups). Following Dose 1, fever ≥38.0° C. was reported by 50.0% (6/12)of BNT162b1 recipients in the 100 μg group and 8.3% (1/12) ofparticipants each in the 10 μg and 30 μg groups. Following Dose 2, 8.3%(1/12) of participants in the 10 μg group and 75.0% (9/12) ofparticipants in the 30 μg group reported fever 38.0° C. No Grade 4systemic events or fever were reported. (FIGS. 35 & 36 ). Most localreactions and systemic events peaked by Day 2 after vaccination andresolved by Day 7. Based on the reactogenicity profile, participants whoreceived an initial 100 μg dose did not receive a second vaccination.

Adverse events were reported by 50.0% (6/12) of participants whoreceived 10 μg or 30 μg of BNT162b1, by 58.3% (7/12) of those whoreceived 100 μg of BNT162b1, and by 11.1% (1/9) of placebo recipients.Two participants reported a severe AE, one at the 30 μg dose level(Grade 3 pyrexia 2 days after vaccination) and one at the 100 μg doselevel (sleep disturbance 1 day after vaccination). Related AEs werereported by 25% (3/12) to 50% (6/12) of BNT162b1 recipients and by 11.1%(1/6) of those who received placebo. No serious adverse events werereported.

No Grade 1 or greater change in routine clinical laboratory values orlaboratory abnormalities were observed for most subjects after either ofthe BNT162b1 vaccinations. The most notable changes were decreases inlymphocyte count in 8.3% (1/12), 45.5% (5/11), and 50.0% (6/12) ofparticipants who received 10 μg, 30 μg, or 100 μg, respectively, ofBNT162b1. One participant each at the 10 μg (8.3%) and 30 μg (9.1%) doselevels and 4 participants at the 100 μg dose level (33.3%) had Grade 3decreases in lymphocytes. These hematological changes, which were notedin blood drawn 1-3 days after Dose 1, returned to normal 6-8 days aftervaccination. None of the changes in laboratory values after vaccinationwere associated with clinical findings. In addition, Grade 2 neutropeniawas noted 6-9 days after the second dose of 10 μg or 30 μg BNT162b1, in1 participant each. The neutrophil count was not repeated for these twosubjects however they continue be followed in the study and no adverseevents or clinical manifestation of neutropenia have been reported todate.

Immunogenicity

RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titers wereassessed in sera drawn at baseline and at 7 and 21 days after the firstdose and 7 days (Day 28) after the second dose of BNT162b1 (FIG. 37 a ).By 21 days after the first dose (for all three dose levels), geometricmean concentrations (GMCs) of RBD-binding IgG were 534-1,778 units/mL,compared to 602 units/mL for a panel of COVID-19 convalescent humansera. By 7 days after the second dose (for the 10 μg and 30 μg doselevels) RBD-binding IgG GMCs had increased to 4,813-30,207 units/mL.Because the participants who received a first dose of 100 μg BNT162b1did not receive a second dose, the development of the antibody responsewithout a second dose could not be evaluated, and there was no furtherincrease in RBD-binding antibody concentration beyond 21 days after thefirst dose among participants in this dosing group. Highly elevatedRBD-binding antibody concentrations persisted to Day 35 (two weeks afterthe second dose) in the participants who received 10 μg and 30 μg doselevels of BNT162b1.

Modest increases in SARS-CoV-2 neutralizing geometric mean titers (GMTs)were observed 21 days after Dose 1 (FIG. 37 b ). Substantially greaterserum neutralizing GMTs were achieved 7 days after participants receiveda second 10 μg or 30 μg dose, reaching 168-267, compared to 94 for theCOVID-19 convalescent serum panel.

Discussion

The RNA-based vaccine candidate BNT162b1 was safe and well tolerated.All dose levels exhibited a tolerability and safety profile consistentwith those previously observed for mRNA-based vaccines. A clear doselevel response was observed after Doses 1 and 2 in adults 18-55 years ofage. Reactogenicity was generally higher after the second dose, butsymptoms resolved within a few days after presentation. Based on thetolerability profile of the first dose at the 100 μg dose level,participants randomized to the 100 μg group did not receive a secondvaccination. Transient decreases in lymphocytes (Grades 1-3) wereobserved within a few days after vaccination; however, lymphocyte countsreturned to baseline within 6-8 days in all participants. Theselaboratory abnormalities were not associated with clinical findings.Lymphopenia following vaccination is most likely explained by transientmigration of lymphocytes into the tissues.

Robust immunogenicity was observed after vaccination with BNT162b1.RBD-binding IgG concentrations were detected at Day 21 and substantiallyincreased 7 days after the booster dose given at Day 21. After the firstdose, the RBD-binding IgG GMCs in vaccinated participants (10 μg doselevel) were similar to those observed in a panel of 38 sera fromCOVID-19 convalescing humans obtained 20-40 days after the onset ofsymptoms and at least 14 days after the start of asymptomaticconvalescence. In sera drawn from the 30 μg and 100 μg dose levelcohorts, GMCs were substantially higher than in the convalescent serumpanel. After the booster vaccinations (Dose 2) with 10 μg or 30 μgBNT162b1, the RBD-binding IgG GMCs were 8.0-fold to 50-fold higher thanthe convalescent serum panel GMC.

Sera from vaccinated participants were also tested in the SARS-CoV-2neutralization assay. Neutralization titers were measurable at Day 21for all dose levels. At Day 28 (7 days after the booster dose),substantial SARS-CoV-2 neutralization titers were observed. The virusneutralizing GMTs after the 10 μg and 30 μg booster vaccinations (Dose2) were, respectively, 1.8-fold and 2.8-fold higher than theneutralizing GMT of the convalescent serum panel. As the 100 μg doselevel cohort was not boosted, no corresponding data for immunogenicityafter a second vaccination are available.

These clinical findings for the BNT162b1 vaccine candidate are veryencouraging and provide strong evidence supporting accelerateddevelopment and at-risk manufacturing to maximize the opportunity forthe soonest availability of a prophylactic vaccine to prevent COVID-19.

Example 7: Concurrent Antibody and, T Cell and Cytokine ResponsesElicited by a COVID-19 RNA Vaccine

In this example, we present characterisation of antibody and T cellresponses after BNT162b1 vaccination from a non-randomized open-labelphase I/II trial in healthy adults, 18-55 years of age. Two doses, of 1μg, 10 μg, 30 μg and 50 μg of BNT162b1 administered 21 days apartelicited concomitant antibody, and robust CD4⁺ and CD8⁺ T cellresponses. All subjects exhibited strong antibody responses with IgGconcentrations significantly above those observed in COVID-19convalescent human sera. Day 43 SARS-CoV-2 serum neutralising geometricmean titers were in the range of 0.7-fold (1 μg) to 3.3-fold (50 μg)compared to those of a panel of COVID-19 convalescent human sera, andwere broadly active against diverse SARS-CoV-2 spike variants.Interferon (IFN)γ, an immune stimulatory cytokine with anti-viralproperties, was produced by a high frequency of RBD-antigen specificCD8⁺ T and numerous CD4⁺ T cells. IL-12p70, which reinforces a T_(H)1immune cell profile, was detected in RBD-stimulated immune cells. Therobust RBD-specific antibody, T-cell and favorable cytokine responses bythe BNT162b1 mRNA vaccine suggests a potential for multiple beneficialprotective mechanisms against COVID-19.

Materials and Methods Clinical Trial Design

Study BNT162-01 (NCT04380701—Germany trial) is an ongoing,first-in-human, Phase I/II, open-label dose-finding clinical trial toassess the safety, tolerability, and immunogenicity of ascending doselevels of various intramuscularly administered BNT162 mRNA vaccinecandidates. Healthy men and non-pregnant women 18 to 55 years (amendedto add 56-85 of age) of age are eligible. Key exclusion criteriaincluded previous clinical or microbiological diagnosis of COVID-19;receipt of medications to prevent COVID-19; previous vaccination withany coronavirus vaccine; a positive serological test for SARS-CoV-2 IgMand/or IgG at the screening visit; and a SARS-CoV-2 NAAT-positive nasalswab within 24 hours before study vaccination; those with increased riskfor severe COVID-19; immunocompromised individuals, those with knowninfection with HIV, hepatitis C virus, or hepatitis B virus and thosewith a history of autoimmune disease. The primary endpoints of the studyare safety and immunogenicity.

In the part of the study reported here five dose levels (1 μg, 10 μg, 30μg, 50 μg or 60 μg) of the BNT162b1 candidate were assessed at one sitein Germany with 12 healthy volunteers per dose level in a doseescalation and de-escalation design. Sentinel dosing was performed ineach dose-escalation cohort. Progression in that cohort and doseescalation required data review by a safety review committee. Subjectsreceived a BNT162b1 prime dose on day 1, and a boost dose on day 22±2.Serum for antibody assays was obtained on day 1 (pre-prime), 8±1(post-prime), 22±2 (pre-boost), 29±3 and 43±4 (post-boost). PBMCs for Tcell studies were obtained on day 1 (pre-prime) and 29±3 (post-boost).One subject of the 10 μg, and one subject of the 50 μg dose cohort leftthe study prior to the boost immunisation due to withdrawal of consentand private reasons.

The presented data comprise the BNT162b1-immunised cohorts only and arebased on a preliminary analysis with a data extraction date of Jul. 13,2020, focused on analysis of vaccine-induced immunogenicity (secondaryendpoint) descriptively summarised at the various time points. Allparticipants with data available were included in the immunogenicityanalyses.

The trial was carried out in Germany in accordance with the Declarationof Helsinki and Good Clinical Practice Guidelines and with approval byan independent ethics committee (Ethik-Kommission of theLandesärztekammer Baden-Württemberg, Stuttgart, Germany) and thecompetent regulatory authority (Paul-Ehrlich Institute, Langen,Germany). All subjects provided written informed consent.

Manufacturing of RNA

BNT162b1 incorporates a Good Manufacturing Practice (GMP)-grade mRNAdrug substance that encodes the trimerized SARS-CoV-2 spike glycoproteinRBD antigen. The RNA is generated from a DNA template by in vitrotranscription in the presence of 1-methylpseudouridine-5′-triphosphate(m1YTP; Thermo Fisher Scientific) instead of uridine-5′-triphosphate(UTP). Capping is performed co-transcriptionally using a trinucleotidecap 1 analogue ((m₂ ^(7,3′-O))Gppp(m^(2′-O))ApG; TriLink). Theantigen-encoding RNA contains sequence elements that increase RNAstability and translation efficiency in human dendritic cells (Holtkamp,S. et al., Blood 108, 4009-4017 (2006); Orlandini von Niessen, A. G. etal., Mol. Ther. 27, 824-836 (2019)). The mRNA is formulated with lipidsto obtain the RNA-LNP drug product. The vaccine was transported andsupplied as a buffered-liquid solution for IM injection and was storedat −80° C.

Proteins and Peptides

A pool of 15-mer peptides overlapping by 11 aa and covering the wholesequence of the BNT162b1-encoded SARS-CoV-2 RBD, was used for ex vivostimulation of PBMCs for flow cytometry, IFNγ ELISpot and cytokineprofiling. CEF (CMV, EBV, influenza virus; HLA class I epitope peptidepool) and CEFT (CMV, EBV, influenza virus, tetanus toxoid; HLA class IIepitope peptide pool) (both JPT Peptide Technologies) were used ascontrols for general T-cell reactivity.

Human Convalescent Sera and PBMC Panel

Human SARS-CoV-2 infection/COVID-19 convalescent sera (n=38) were drawnfrom subjects 18-83 years of age at least 14 days after PCR-confirmeddiagnosis and at a time when the subjects were asymptomatic. Serumdonors had symptomatic infections (n=35), or had been hospitalized(n=1). Sera were obtained from Sanguine Biosciences (Sherman Oaks,Calif.), the MT Group (Van Nuys, Calif.) and Pfizer Occupational Healthand Wellness (Pearl River, N.Y.). Human SARS-CoV-2 infection/COVID-19convalescent PBMC samples (n=6) were collected from subjects 41-79 yearsof age 45-59 days after PCR-confirmed diagnosis when subjects wereasymptomatic. PBMC donors had asymptomatic/mild infections (n=4;clinical score 1 and 2) or had been hospitalized (n=2; clinical score 4and 5). Blood samples were obtained from the Frankfurt UniversityHospital (Germany).

Cell Culture and Primary Cell Isolation

Vero cells (ATCC CCL-81) and Vero E6 cells (ATCC CRL-1586) were culturedin Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco)supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). Celllines were tested for mycoplasma contamination after receipt and beforeexpansion and cryopreservation. Peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll-Hypaque (Amersham Biosciences) densitygradient centrifugation and cryopreserved prior to subsequent analysis.

RBD Binding IgG Antibody Assay

A recombinant SARS-CoV-2 RBD containing a C-terminal Avitag™ (AcroBiosystems) was bound to streptavidin-coated Luminex microspheres.Heat-inactivated subject sera were diluted 1:500, 1:5,000, and 1:50,000.Following an overnight incubation at 2-8° C. while shaking, plates werewashed in a solution containing 0.05% Tween-20. A secondaryfluorescently labelled goat anti-human polyclonal antibody (JacksonLabs) was added for 90 minutes at room temperature while shaking, beforeplates were washed once more in a solution containing 0.05% Tween-20.Data were captured as median fluorescent intensities (MFIs) using aLuminex reader and converted to U/mL antibody concentrations using areference standard curve with arbitrary assigned concentrations of 100U/mL and accounting for the serum dilution factor. Three dilutions areused to increase the likelihood that at least one result for any samplewill fall within the useable range of the standard curve. Assay resultswere reported in U/mL of IgG. The final assay results are expressed asthe geometric mean concentration of all sample dilutions that produced avalid assay result within the assay range.

SARS-CoV-2 Neutralisation Assay

The neutralisation assay used a previously described strain ofSARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics andengineered by the insertion of an mNeonGreen (mNG) gene into openreading frame 7 of the viral genome (Xie, X. et al., Cell Host Microbe27, 841-848.e3 (2020)). This reporter virus generates similar plaquemorphologies and indistinguishable growth curves from wild-type virus.Viral master stocks (2×10⁷ PFU/mL) were grown in Vero E6 cells aspreviously described (Xie, X. et al., Cell Host Microbe 27, 841-848.e3(2020)). Serial dilutions of heat-inactivated sera were incubated withthe reporter virus (2×10⁴ PFU per well for a final multiplicity ofinfection (MOI) of 0.5 to yield approximately a 10-30% infection rate ofthe Vero monolayer) for 1 hour at 37° C. before inoculating Vero CCL81cell monolayers (targeted to have 8,000 to 15,000 cells per well) in96-well plates to allow accurate quantification of infected cells. Totalcell counts per well were enumerated by nuclear stain (Hoechst 33342)and fluorescent virally infected foci were detected 16-24 hours afterinoculation with a Cytation 7 Cell Imaging Multi-Mode Reader (Biotek)with Gen5 Image Prime version 3.09. Titers were calculated in GraphPadPrism version 8.4.2 by generating a 4-parameter (4PL) logistical fit ofthe percent neutralisation at each serial serum dilution. The 50%neutralisation titre (VNT₅₀) was reported as the interpolated reciprocalof the dilution yielding a 50% reduction in fluorescent viral foci.

VSV-SARS-CoV-2 Spike Variant Pseudovirus Neutralisation Assay

VSV-SARS-CoV-2-S pseudoparticle generation and neutralisation assayswere performed as previously described (Baum, A. et al., Science,eabd0831 (2020). doi:10.1126/science.abd0831). Briefly, human codonoptimized SARS-CoV-2 spike (GenBank: MN908947.3) was synthesised(Genscript) and cloned into an expression plasmid. SARS-CoV-2 completegenome sequences were downloaded from GISAID Nucleotide database(www.gisaid.org) (last accessed 24 Aug. 2020). Sequences were curatedand genetic diversity of the Spike-encoding gene was assessed acrosshigh quality genome sequences using custom pipelines. Amino acidsubstitutions were cloned into the spike expression plasmid usingsite-directed mutagenesis. HEK293T cells (ATCC CRL-3216) were seeded(culture medium: DMEM high glucose (Life Technologies) supplemented with10% heat-inactivated fetal bovine serum (FBS; Life Technologies) andPenicillin/Streptomycin/L-Glutamine (Life Technologies)) and transfectedthe following day with spike expression plasmid using Lipofectamine LTX(Life Technologies) following the manufacturer's protocol. At 24 hourspost-transfection at 37° C., cells were infected with theVSVΔG:mNeon/VSV-G virus diluted in Opti-MEM (Life Technologies) at amultiplicity of infection of 1. Cells were incubated 1 hour at 37° C.,washed to remove residual input virus and overlaid with infection medium(DMEM high glucose supplemented with 0.7% Low IgG BSA (Sigma), sodiumpyruvate (Life Technologies) and 0.5% Gentamicin (Life Technologies)).After 24 hours at 37° C., the supernatant containing VSV-SARS-CoV-2-Spseudoparticles was collected, centrifuged at 3000×g for 5 minutes toclarify and stored at −80° C. until further use.

For pseudovirus neutralisation assays, Vero cells (ATCC CCL-81) wereseeded in 96-well plates in culture medium and allowed to reachapproximately 85% confluence before use in the assay (24 hours later).Sera were serially diluted 1:2 in infection medium starting with a 1:40dilution. VSV-SARS-CoV-2-S pseudoparticles were diluted 1:1 in infectionmedium for a fluorescent focus unit (ffu) count in the assay of^(˜)1000. Serum dilutions were mixed 1:1 with pseudoparticles for 30minutes at room temperature prior to addition to Vero cells andincubation at 37° C. for 24 hours. Supernatants were removed andreplaced with PBS (Gibco), and fluorescent foci were quantified usingthe SpectraMax i3 plate reader with MiniMax imaging cytometer (MolecularDevices). Neutralisation titers were calculated in GraphPad Prismversion 8.4.2 by generating a 4-parameter logistical (4PL) fit of thepercent neutralisation at each serial serum dilution. The 50%pseudovirus neutralisation titre (pVNT₅₀) was reported as theinterpolated reciprocal of the dilution yielding a 50% reduction influorescent viral foci.

IFNγ ELISpot.

IFNγ ELISpot analysis was performed ex vivo (without further in vitroculturing for expansion) using PBMCs depleted of CD4⁺ and enriched forCD8⁺ T cells (CD8⁺ effectors), or depleted of CD8⁺ and enriched for CD4⁺T cells (CD4⁺ effectors). Tests were performed in duplicate and with apositive control (anti-CD3 monoclonal antibody CD3-2 (1:1,000;Mabtech)). Multiscreen filter plates (Merck Millipore) pre-coated withIFNγ-specific antibodies (ELISpotPro kit, Mabtech) were washed with PBSand blocked with X-VIVO 15 medium (Lonza) containing 2% human serumalbumin (CSL-Behring) for 1-5 hours. Per well, 3.3×10⁵ effector cellswere stimulated for 16-20 hours with an overlapping peptide poolrepresenting the vaccine-encoded RBD. Bound IFNγ was visualized using asecondary antibody directly conjugated with alkaline phosphatasefollowed by incubation with BCIP/NBT substrate (ELISpotPro kit,Mabtech). Plates were scanned using an AID Classic Robot ELISPOT Readerand analysed by ImmunoCapture V6.3 (Cellular Technology Limited) or AIDELISPOT 7.0 software (AID Autoimmun Diagnostika). Spot counts weredisplayed as mean values of each duplicate. T-cell responses stimulatedby peptides were compared to effectors incubated with medium only asnegative control using an in-house ELISpot data analysis tool (EDA),based on two statistical tests (distribution-free resampling) accordingto Moodie et al. (Moodie, Z., et al., J. Immunol. Methods 315, 121-32(2006); Moodie, Z. et al., Cancer Immunol. Immunother. 59, 1489-501(2010)), to provide sensitivity while maintaining control over falsepositives.

To account for varying sample quality reflected in the number of spotsin response to anti-CD3 antibody stimulation, a normalisation method wasapplied to enable direct comparison of spot counts/strength of responsebetween individuals. This dependency was modelled in a log-linearfashion with a Bayesian model including a noise component (unpublished).For a robust normalization, each normalisation was sampled 1000 timesfrom the model and the median taken as normalized spot count value.Likelihood of the model: log λ_(E)=α log λ_(P)+log β_(j)+σε, where λ_(E)is the normalized spot count of the sample, a is a stable factor(normally distributed) common among all positive controls λ_(P), β_(j) asample j specific component (normally distributed) and σε is the noisecomponent, of which σ is Cauchy distributed and E is Student's-tdistributed. β_(j) ensures that each sample is treated as a differentbatch.

Flow Cytometry

Cytokine-producing T cells were identified by intracellular cytokinestaining. PBMCs thawed and rested for 4 hours in OpTmizer mediumsupplemented with 2 μg/mL DNAsel (Roche), were restimulated with apeptide pool representing the vaccine-encoded SARS-CoV-2 RBD (2μg/mL/peptide; JPT Peptide Technologies) in the presence of GolgiPlug(BD) for 18 hours at 37° C. Controls were treated with DMSO-containingmedium. Cells were stained for viability and surface markers in flowbuffer ((DPBS (Gibco) supplemented with 2% FCS (Biochrom), 2 mM EDTA(Sigma-Aldrich)) for 20 minutes at 4° C. Afterwards, samples were fixedand permeabilized using the Cytofix/Cytoperm kit according tomanufacturer's instructions (BD Biosciences). Intracellular staining wasperformed in Perm/Wash buffer for 30 minutes at 4° C. Samples wereacquired on a FACS VERSE instrument (BD Biosciences) and analysed withFlowJo software version 10.5.3 (FlowJo LLC, BD Biosciences).RBD-specific cytokine production was corrected for background bysubtraction of values obtained with DMSO-containing medium. Negativevalues were set to zero. Cytokine production in FIG. 42 b was calculatedby summing up the fractions of all CD4⁺ T cells positive for eitherIFNγ, IL-2 or IL-4, setting this sum to 100% and calculating thefraction of each specific cytokine-producing subset thereof.

Cytokine Profiling

Human PBMCs were restimulated for 48 hours with SARS-CoV-2 RBD peptidepool (2 μg/mL final concentration per peptide). Stimulation withDMSO-containing medium served as negative controls. Concentrations ofTNF, IL-1β and IL-12p70 in supernatants were determined using abead-based, 11-plex T_(H)1/T_(H)2 human ProcartaPlex immunoassay (ThermoFisher Scientific) according to the manufacturer's instructions.Fluorescence was measured with a Bioplex200 system (Bio-Rad) andanalysed with ProcartaPlex Analyst 1.0 software (Thermo FisherScientific). RBD-specific cytokine production was corrected forbackground by subtraction of values obtained with DMSO-containingmedium. Negative values were set to zero.

Results Study Design and Analysis Set

Between Apr. 23, 2020 and May 22, 2020, 60 subjects were vaccinated withBNT162b1. Twelve participants per 1 μg, 10 μg, 30 μg, and 50 μg doselevels received a first dose on day 1 and were boosted on day 22, and 12participants received a 60 μg prime dose on Day 1 only (FIG. 43 ). Thestudy population consisted of healthy males and non-pregnant femaleswith a mean age of 41 years (range 19 to 55 years) with equal genderdistribution. Most participants were Caucasian (96.7%) with one AfricanAmerican and one Asian subject (1.7% each). Preliminary data analysiswas focused on immunogenicity (Table 4).

TABLE 4 Subject disposition and analysis sets. T-cell Antibody analysisanalysis BNT162b1 Day Day Day Day Day Day Day Cohort Prime Boost 1 8 ± 122 ± 2 29 ± 3 43 ± 4 1 29 ± 3  1 μg 12 12 12 12 12 12 12 8 8 10 μg 12 1112 12 12 11 11 (10) 10 (6) 10 (6) 30 μg 12 12 12 12 12 12 12 (10) 10 (7)10 (7) 50 μg 12 11 12 12 12 11  6  8 (5)  8 (5) 60 μg 12 N/A 12 12 11 12N/A N/A N/A Antibody analysis: Values indicated number of subjects forwhich virus neutralisation assay was performed. Values in parenthesesindicate number of subjects for which RBD binding IgG antibody assay wasperformed. T-cell analysis: Values indicated number of subjects forwhich IFNγ ELISpot was performed. Values in parentheses indicate numberof subjects for which flow cytometry was performed. N/A: Samples not yetavailable.

Briefly, no serious adverse events (SAE), no unexpected toxicities, andno withdrawals due to related AEs were observed. Most reported solicitedAEs were signs and symptoms of vaccine reactogenicity, typically withonset within the first 24 hours post immunisation, such as systemic, andinjection site reactions, chiefly symptoms of pain and tenderness (FIG.44 ). Symptomatology was mostly mild or moderate in intensity withoccasional severe (Grade 3) AEs such as fever, chills, headache, muscleand joint pain, and injection site reactions. All AEs resolvedspontaneously, mostly within 24 hours of onset and could be managed withsimple measures (e.g. paracetamol). Based on the reactogenicity reportedafter the first dose, participants who had received an initial 60 μgdose did not receive a second 60 μg dose. Whereas no relevant change inroutine clinical laboratory values occurred after BNT162b1 vaccination,a transient increase of the inflammatory marker C-reactive protein (CRP)and temporary reduction of blood lymphocyte counts were observed in adose-dependent manner in vaccinated subjects (FIG. 45 ). Based on ourprevious clinical experience with RNA vaccines, the latter is likelyattributable to innate immune stimulation-related transientredistribution of lymphocytes (Kamphuis, E., et al., Blood 108, 3253-61(2006)).

Vaccine-Induced Antibody Response

RBD-binding IgG concentrations and SARS-CoV-2 neutralising titers wereassessed at baseline, 7 and 21 days after the BNT162b1 prime dose (days8 and 22), and 7 and 21 days after the boost dose (days 29 and 43),except for the 60 μg cohort, which received prime only (FIG. 39 ).

All subjects including those who received the 1 μg dose showed a strong,dose-dependent vaccine-induced antibody response. Twenty-one days afterthe priming dose (for the four dose levels ranging from 1-50 μg),geometric mean concentrations (GMCs) of RBD-binding IgG were dosedependently about 265-1,672 U/mL (FIG. 39 ). Seven days after theboosting dose (day 29) RBD-binding IgG GMCs in subjects treated with1-50 μg BNT162b1 strongly increased dose-dependently to about2,015-25,006 U/mL. At day 43 (21 days after boost), RBD-binding antibodyGMCs were in the range of about 3,920-22,700 U/mL in BNT162b1 vaccinatedindividuals as compared to about 602 U/mL measured in a panel of serafrom 38 SARS-CoV-2 infection convalescent patients (18-83 years of age)drawn at least 14 days after PCR-confirmed diagnosis. In the 60 μg dosecohort with prime dose only, RBD-binding IgG GMCs were about 1,058 U/mLby day 29 indicating the necessity of the second dose for boosting theantibody titer.

SARS-CoV-2 neutralising antibody geometric mean titers (GMTs) increasedmodestly in a dose-dependent manner 21 days after the priming dose (FIG.40 a ). Substantially higher serum neutralizing GMTs were achieved 7days after the boost dose, reaching about 36 (1 μg dose level), about158 (10 μg dose level), about 308 (30 μg dose level), and about 578 (50μg dose level), compared to about 94 for the convalescent serum panel.On day 43 (21 days after the boost), depending on the dose level, theneutralising antibody GMT were further increased to about 62 (1 μgdose), were relatively stable at about 126 (10 μg dose), or decreasedslightly to about 157 (30 μg dose), and about 309 (50 μg dose).Neutralising antibody GMTs were strongly correlated with RBD-binding IgGGMC (FIG. 40 b ). In summary, neutralising antibody titers were largelyin the range of those previously reported in the US study with BNT162b1.Further, by 7 days after the second dose, sera of vaccinated subjectsdisplayed broad neutralising activity across a panel of seventeenSARS-CoV-2 spike variants identified in publicly available SARS-CoV-2sequences, including sixteen RBD mutants (Baum, A. et al., Science,eabd0831 (2020). doi:10.1126/science.abd0831) and the dominant spikevariant D614G (Baum, A. et al., Science, eabd0831 (2020).doi:10.1126/science.abd0831) (FIG. 40 c ).

Vaccine-Induced T Cell Responses

CD4⁺ and CD8⁺ T cell responses in BNT162b1 immunized subjects werecharacterized prior to prime vaccination (day 1) and on day 29 afterprime (7 days after boost vaccination) using direct ex vivo IFNγ ELISPOTwith PBMCs from 36 subjects across the 1 μg to 50 μg dose cohorts (FIG.41 ). In this assay, CD4⁺ or CD8⁺ T cell effectors were stimulatedovernight with overlapping peptides representing the full-lengthsequence of the vaccine-encoded RBD.

Of 36 subjects, 34 (94.4%, including all subjects treated with ≥10 μgBNT162b1) mounted RBD-specific CD4⁺ T cell responses. The magnitudevaried between individuals with the strongest CD4 T cell responses beingmore than 10-fold of the memory responses observed against a panel ofcytomegalovirus (CMV), Epstein Barrvirus (EBV), influenza virus andtetanus toxoid-derived immuno-dominant peptides in the same subjects(FIG. 41 a-c ). No CD4⁺ T cell responses were detectable at baseline,except for one subject with a low number of preexisting RBD-reactiveCD4⁺ T cells, which increased significantly after vaccination(normalized mean spot count from 63 to 1,519, in the 50 μg dose cohort).The strength of RBD-specific CD4⁺ T cell responses correlated positivelywith both RBD-binding IgG and with SARS-CoV-2 neutralising antibodytiters (FIG. 41 d , FIG. 46 a ), in line with the concept ofintramolecular help (Sette, A. et al., Immunity 28, 847-58 (2008)). Thetwo subjects lacking CD4⁺ response had no detectable VNT₅₀ titers either(FIG. 41 d ).

Vaccine-induced CD8⁺ T cell responses, some strong ones were mounted bythe majority of subjects (29/36, 80.6%) (FIG. 41 a ) and were quitecomparable with memory responses against CMV, EBV, Influenza virus andtetanus toxoid in the same subjects (FIG. 41 b, c ). The strength ofRBD-specific CD8⁺ T cell responses correlated positively withvaccine-induced CD4⁺ T cell responses but did not significantlycorrelate with SARS-CoV-2 neutralizing antibody titers (FIG. 46 b, c ).

Of note, although at 1 μg BNT162b1 the immunogenicity rate was lower(6/8 responding subjects), the magnitude of vaccine-induced CD4⁺ andCD8⁺ T cells in some subjects was almost as high as with 50 μg BNT162b1(FIG. 41 a ). To assess functionality and polarization of RBD-specific Tcells, cytokines secreted in response to stimulation with the vaccineantigen were determined by intracellular staining (ICS) with IFNγ, IL-2and IL-4 specific antibodies in pre- and post-vaccination PBMCs of 18BNT162b1 immunised subjects. RBD-specific CD4⁺ T cells secreted IFNγ,IL-2, or both, but did not secrete IL-4 (FIG. 42 a-c ). Similarly, afraction of RBD-specific IFNγ+CD8⁺ T cells also secreted IL-2.

The mean fraction of RBD-specific T cells within total circulating Tcells obtained by BNT162b1 vaccination was substantially higher thanthat observed in six subjects recovered from COVID-19. Frequency ofRBD-specific IFNγ⁺ CD8⁺ T cells reached up to several percent of totalperipheral blood CD8⁺ T cells (FIG. 42 c ). Analysis of supernatants ofPBMCs stimulated ex vivo with overlapping RBD peptides from a subgroupof five vaccinated subjects showed cognate release of proinflammatorycytokines TNF, IL-1β and IL-12p70 (FIG. 42 d ). In summary, thesefindings indicate that BNT162b1 induces functional and proinflammatoryCD4⁺/CD8⁺ T cell responses in almost all subjects, with T_(H)1 polarizedhelper response.

Discussion

We observed concurrent production of neutralising antibodies, activationof virus-specific CD4⁺ and CD8⁺ T cells, and robust release ofimmune-modulatory cytokines such as IFNγ, which represents a coordinatedimmune response to counter a viral intrusion (for review Vabret, N. etal., Immunity 52, 910-941 (2020)). IFNγ represents a key cytokine forseveral anti-viral responses. Indeed, patients with IFNγ genepolymorphism related to impaired IFNγ activity have been shown todisplay 5-fold increased susceptibility to SARS (Chong, W. P. et al.,BMC Infect. Dis. 6, 82 (2006)). Also, IFNγ acts in synergy with type Iinterferons to inhibit replication of SARS-CoV-2 (Sainz, B., et al.,Virology 329, 11-7 (2004)). The robust production of IFNγ from CD8⁺ Tcells indicates a favourable immune response with both anti-viral andimmune-augmenting properties.

Importantly, the detection of IFNγ, IL-2 and IL-12p70 but not IL-4indicates a favorable T_(H)1 profile and the absence of a potentiallydeleterious T_(H)2 immune response. CD4⁺ and CD8⁺ T cells may conferlong lasting immunity against corona viruses as indicated in SARS-CoV-1survivors, where CD8⁺ T-cell immunity persisted for 6-11 years (Vabret,N. et al., Immunity 52, 910-941 (2020); Ng, O.-W. et al., Vaccine 34,2008-14 (2016)).

Some cases of asymptomatic virus exposure have been associated withcellular immune response without seroconversion indicating thatSARS-Cov-2 specific T cells could be relevant in disease control even inthe absence of neutralising antibodies (Gallais, F. et al. (2020).doi:medRxiv: 10.1101/2020.06.21.20132449). Almost all vaccinatedvolunteers mounted RBD-specific T cell responses detected with an exvivo ELISpot assay, which was performed without prior expansion of Tcells that captures only high-magnitude T cell responses. Although thestrength of the T-cell responses varied considerably between subjects,we observed no clear dose dependency of the T-cell response strength inthe dose range of 1 μg to 50 μg, indicating that stimulation and robustexpansion of T cells might be accomplished at the lowest mRNA-encodedimmunogen levels.

The study confirms the dose-dependency of RBD-binding IgG andneutralisation responses, reproduces our previous findings for 10 and 30μg dose levels in the US trial, and shows that neutralising antibodytiters are further increased by a prime/boost regimen at 50 μg.

A notable observation is that two injections of BNT162b1 at a dose levelas low as 1 μg are capable of inducing RBD-binding IgG levels higherthan those observed in convalescent sera, and serum neutralisingantibody titers that are still increasing up to day 43. Considering thatthe magnitude of a protective neutralising antibody titer is not known,and given the substantial T-cell responses we observed for some subjectsin the 1 μg cohort may hold the promise that a considerable fraction ofindividuals may benefit even from this lowest tested dose level.

A purely RBD-directed immunity might be considered prone to escape ofthe virus by single amino acid changes in this small domain. However,neutralisation of 17 pseudo-typed viruses, 16 of which enter cells usinga spike with a different RBD variant found in circulating strains andone of which uses the dominant spike variant D614G, alleviates thispotential concern.

Example 8: Summary of Safety and Immunogenicity Data from Stage 1 ofCOVID-19 Vaccine BNT162

This example provides additional safety and immunogenicity data for theBNT162b1 and BNT162b2 vaccine candidates. These safety and tolerabilitydata, as well as immunoglobulin G (IgG) binding and severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2) neutralization titerdata, are coming from US participants in a Stage 1 US Study of thesevaccine candidates.

For BNT162b1, the following is observed:

For 10 μg to 30 μg dose levels, reactogenicity (particularly systemicevents) increases with increasing dose level in 18 to 55-year-old and 65to 85-year-old participants. Reactogenicity (particularly systemicevents) increased after Dose 2 compared to Dose 1.

For BNT162b2, the following is observed:

Dose level- and dose number-dependent increases in reactogenicity wereminimal to modest in either age group. Based on all available data, thereactogenicity profile observed with BNT162b2 (and particularly SEQ IDNO:20) is quite favorable.

The immunogenicity data presented herein, focusing on SARS-CoV-2neutralizing responses post-dose 2, allow to conclude the following:

For BNT162b1 at Day 28 (7 days post-dose 2):

Neutralizing antibody responses elicited after immunization with 10 μgand 30 μg dosages (where data are available in both age groups) arehigher in adults 18 to 55 years of age compared to the 65 to 85-year-oldgroup. In the 65 to 85 year old group, neutralizing antibody responsesafter 20 μg and 30 μg dosages were similar, although numerically higherat the 20 μg dose level.

For BNT162b2 (particularly, SEQ ID NO:20) at Day 28 (7 days post-dose2):

Neutralizing antibody responses after the 20 μg dosage (where data areavailable in both age groups) were higher in the 18 to 55 year old groupcompared to the 65 to 85-year-old group. In the 18 to 55 year-old group,neutralizing antibody responses were higher after receiving 20 μgcompared to 10 μg dose levels. The S1 IgG binding antibody data in FIG.56 , and comparisons of post-dose 1 responses across dose levels thatare highest at the 30 μg dose level, suggest that neutralizing antibodylevels will likely also be higher for the 30 μg dose level post-dose 2,as levels of binding antibody correlate well with neutralizing antibodylevels. In the 65 to 85 year old group, neutralizing antibody responsesafter 20 μg and 30 μg doses were higher at the 30 μg dose level.

The data overall show similar neutralizing antibody responses post-dose2 between BNT162b1 and BNT162b2.

Safety and Tolerability of BNT162b1 18-55 Years of Age Groups

Safety data are available for this age group through post-dose 2 for alldose levels, except for 20 μg, which for now has partial data availablethrough post-dose 2 (and 100 μg where a second dose has not beenadministered at the recommendation of the Internal Review Committee(IRC)). Local reactions are shown in FIG. 48 . Systemic events are shownin FIG. 49 .

Immunogenicity of BNT162b1 65-85 Years of Age Groups

Immunogenicity data are available for this age group through post-dose 2for all dose levels. RBD-binding IgG geometric mean concentrations(GMCs) are shown in FIG. 50 . SARS-CoV-2 neutralizing geometric meantiters (GMTs) are shown in FIG. 51 .

Safety and Tolerability of BNT162b2 18-55 Years of Age Groups

Safety data are available for this age group through post-dose 2 for alldose levels. Local reactions are shown in FIG. 52 . Systemic events areshown in FIG. 53 .

65-85 Years of Age Groups

Safety data are available for this age group through post-dose 2 for alldose levels, however, the data for the 10 μg dose level are onlypartial. Local reactions are shown in FIG. 54 . Systemic events areshown in FIG. 55 .

Immunogenicity of BNT162b2 18-55 Years of Age Groups

Immunogenicity data are available for this age group through post-dose 1for the 30 μg dose level and post-dose 2 for the 10 μg and 20 μg doselevels. S1-binding IgG GMCs are shown in FIG. 56 . SARS-CoV-2neutralizing GMTs are shown in FIG. 57 .

65-85 Years of Age Groups

Immunogenicity data are available for this age group through post-dose 2for the 20 μg and 30 μg dose levels. S1-binding IgG GMCs are shown inFIG. 58 . SARS-CoV-2 neutralizing GMTs are shown in FIG. 59 .

Conclusions

The local tolerability profiles of BNT162b1 and BNT162b2 (andparticularly SEQ ID NO:20) and the immune response data are similarbetween the 2 candidates. BNT162b2 (particularly SEQ ID NO:20) may showa favorable systemic reactogenicity profile (particularly in the 65 to85-year-old group). When selecting the dose level for BNT162b2(particularly SEQ ID NO:20), the SARS-CoV-2 neutralizing antibodyresponse level in the 65 to 85-year-old group could be of weight tomaximize the neutralizing antibody responses in this age group, which isat highest risk of severe disease. Comparing the neutralizing antibodylevels in the 20 μg and 30 μg older adult cohorts in this study, the 30μg dose level showed higher neutralizing antibody levels than those inthe 20 μg cohort (FIG. 59 ). In comparison to the neutralizing antibodylevel of a human convalescent serum panel (HCS) with a GMT of 94, theGMT at the 30 μg dose level was 1.6 times the GMT of the HCS; the GMT atthe 20 μg dose level was 0.9 times the GMT of HCS. Thus, both showedneutralizing antibody titres at least comparable to that of the humanconvalescent serum panel. The 38 human SARS-CoV-2 infection/COVID-19convalescent sera were drawn from participants 18 to 83 years of age, atleast 14 days after PCR-confirmed diagnosis, and at a time whenparticipants were asymptomatic. The serum donors predominantly hadsymptomatic infections (35/38), and one had been hospitalized. The serawere obtained from Sanguine Biosciences (Sherman Oaks, Calif.), the MTGroup (Van Nuys, Calif.), and Pfizer Occupational Health and Wellness(Pearl River, N.Y.). In addition, S1-IgG antibody binding concentrationsin both older (FIG. 58 , post-dose 2) and younger (FIG. 56 ,post-dose 1) adult cohorts also favored the selection of the 30 μg doselevel. Preliminary human T cell data that are being generated in aGerman trial with BNT162b2 are confirming the robust CD4⁺ and CD8⁺expected for the RNA platform. With these considerations, it is proposedto use BNT162b2 (particularly SEQ ID NO:20) at the 30 μg dose level toproceed into Phase 2b/3 because this dose and construct provides theoptimum combination of a favorable reactogenicity profile and a robustimmune response, likely to afford protection against COVID-19 in youngerand older adults.

Example 9: Immunology of COVID-19 Vaccine BNT162

To support progression to Phase 2/3 in adults 18-85 years of age,provided herein are nonclinical and clinical data summarizing the T cellresponse following BNT162b2 immunization in mice and in humans enrolledin a trial with BNT162. The following immunogenicity data are provided:

1. Preliminary and unaudited mouse immunogenicity data: IFNγ ELISpot(FIG. 60 ), intracellular cytokine staining, and Luminex quantificationof cytokines produced following BNT162b2 immunization.2. From a German trial (BNT162-01): IFNγ ELISpot (FIG. 61 , FIG. 62 ,FIG. 63 ) for BNT162b2 at the 10 μg dose level in 18-55 year oldparticipants before the first dose and 7 days after dose 2.

T Cell Responses for BNT162b2 in Mice

Four groups of eight female BALB/c mice were immunized on day 0 withdoses of 0.2 μg, 1 μg or 5 μg per animal BNT162b2 (particularly SEQ IDNO:20), or with the buffer alone (control group), by intramuscular (IM)injection. On days 12 and 28, spleens were collected for splenocyteisolation and analysis of T-cell responses using IFNγ ELISpot assays.Luminex assays and intracellular cytokine staining (ICS) were performedto assess cytokine responses. A high fraction of splenocytes of bothCD4+ and CD8+ T-cell phenotypes isolated from BNT162b2-immunized mice ondays 12 and 28 after immunization, when re-stimulated ex vivo with afull-length S peptide mix, exerted a strong antigen-specific IFNγ- andIL-2-response in ELISpot and flow cytometry assays (FIGS. 60 a and b ).Splenocytes harvested on day 28 and stimulated with the full-length Speptide pool produced high levels of the T_(H)1 cytokines IL-2 and IFNγwith correspondingly minimal levels of the T_(H)2 cytokines IL-4, IL-5,and IL-13 in multiplex immunoassays (FIG. 60 c ).

T Cell Responses in Humans for BNT162b2 from German Study

To evaluate the T cell phenotype elicited by immunization of humans withBNT162b2 (particularly SEQ ID NO:20), IFNγ ELISpot was performed onperipheral blood mononuclear cells (PBMCs) obtained from participants ina German study.

IFNγ ELISpot

Vaccine-elicited T cell responses were determined using CD4- orCD8-depleted PBMCs obtained from subjects prior to dose 1 and on day 29(7 days after dose 2). IFNγ ELISpot data were generated for 5 subjectsimmunized with 10 μg of BNT162b2 (particularly SEQ ID NO:20) at day 1and 22. Post-vaccination spike-specific ex vivo CD4+ and CD8+ T cellresponses were detected in 5/5 (100%) subjects, respectively. Allresponses were minimal or undetectable in the prevaccination samples.The responses are considered vaccine induced (FIG. 61 , FIG. 62 , FIG.63 ).

The BNT162b2 vaccine-elicited, antigen specific CD8+ and CD4+ T cellresponses stimulated by S peptide pool 1 (N-terminal portion of thespike, which includes the receptor binding domain [RBD]) and S peptidepool 2 (C-terminal portion of the spike) were comparable to or higherthan the memory responses in the same subjects against CMV, EBV,influenza virus, and tetanus toxoid (FIG. 63 ).

Conclusions

These data for the BNT162b2 vaccine candidate confirm prior resultsobtained in preclinical models and in humans immunized with modRNA(nucleoside-modified) platforms. The data indicate that modRNA elicitssubstantial Th1-type CD4+ and CD8+ T cell responses.

Example 10: A Trimeric SARS-CoV-2 Receptor-Binding Domain RNA Vaccine isHighly Immunogenic and Protective in Non-Human Primates

Here, we report the design and non-clinical development of the BNT162b1vaccine candidate. We demonstrate that nucleoside-modified mRNA encodinga structurally stable, trimerised receptor-binding domain (RBD) ofSARS-CoV-2, encapsulated in lipid nanoparticles (LNP) for effectiveintramuscular delivery, elicits strong antibody and T_(H)1-dominatedcellular immune responses in mice. Immunisation of mice with singledoses of BNT162b1 elicited substantial dose level-dependent increases inpseudovirus neutralisation titers and strong IFNγ-positive CD4+ and CD8+T-cell responses. Prime-boost vaccination of Rhesus macaques withBNT162b1 elicited authentic SARS-CoV-2 neutralising geometric meantiters, 2.6 to 6.0 times those of a SARS-CoV-2 convalescent human serumpanel. Upon SARS-CoV-2 infectious challenge, the immunised macaques hadeither no or more transient presence of viral RNA in the nose and lungsthan did non-immunised control macaques.

Materials and Methods Ethics Statement.

All mouse studies were performed at BioNTech SE, and protocols wereapproved by the local authorities (local welfare committee), conductedaccording to FELASA recommendations and in compliance with the GermanAnimal Welfare Act and Directive 2010/63/EU. Only animals with anunobjectionable health status were selected for testing procedures.

Immunisations for the non-human primate (NHP) study were performed atthe University of Louisiana at Lafayette-New Iberia Research Center(NIRC), which is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC, Animal Assurance #:000452). The work was in accordance with USDA Animal Welfare Act andRegulations and the NIH Guidelines for Research Involving RecombinantDNA Molecules, and Biosafety in Microbiological and BiomedicalLaboratories. All procedures performed on these animals were inaccordance with regulations and established guidelines and were reviewedand approved by an Institutional Animal Care and Use Committee orthrough an ethical review process. Infectious SARS-CoV-2 challenge forthe NHP study was performed at the Southwest National Primate ResearchCenter. Animal husbandry followed standards recommended by AAALACInternational and the NIH Guide for the Care of Use of LaboratoryAnimals. This study was approved by the Texas Biomedical ResearchInstitute Animal Care and Use Committee.

Protein and Peptide Reagents.

A purified recombinant SARS-CoV-2 RBD fusion with a mouse IgG1 constantregion was used as a target for Western Blot and tagged with a humanFc-tag (both Sino Biological) was used in ELISA to detect SARS-CoV-2S-specific IgG. A purified recombinant RBD with a histidine tag (SinoBiological) was used for surface plasmon resonance (SPR) spectroscopy.An overlapping 15-mer peptide pool of the S protein was used forELISpot, cytokine profiling and intracellular cytokine staining. Apeptide control (SPSYVYHQF (SEQ ID NO: 35), derived from gp70 AH-1(Slansky, J. E. et al., Immunity 13, 529-538, 2000)) was used as controlfor ELISpot assays. All peptides were obtained from JPT PeptideTechnologies.

Human Convalescent Sera.

Human COVID-19 convalescent sera (n=38) were drawn from donors 18-83years of age at least 14 days after PCR-confirmed diagnosis and at atime when the participants were asymptomatic. Serum donors hadsymptomatic infections (35/38), or had had been hospitalised (1/38).Sera were obtained from Sanguine Biosciences (Sherman Oaks, Calif.), theMT group (Van Nuys, Calif.) and Pfizer Occupational Health and Wellness(Pearl River, N.Y.).

Cell culture.

Human embryonic kidney (HEK)293T/17 and Vero-76 cells (both ATCC) werecultured in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX™(Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich). Celllines were tested for mycoplasma contamination after receipt, beforeexpansion and cryopreservation. Vero E6 and Vero CCL81 (both ATCC) cellswere cultured in DMEM (Gibco) containing 2% HyClone fetal bovine serumand 100 U/mL penicillium/streptomycin (Gibco). Expi293F™ cells weregrown in Expi293™ media and transiently transfected usingExpiFectamine™293 (all from Thermo Fisher Scientific).

Manufacturing of In Vitro Transcribed RNA.

To generate the template for RNA synthesis, a DNA fragment encoding afusion protein composed of the signal peptide (SP, amino acids 1-16),the SARS-CoV-2 S RBD (GenBank: MN908947) and a T4 fibritintrimerisationmotif (‘foldon’), was cloned into a starting plasmid vector withbackbone sequence elements for improved RNA stability and translationalefficiency (Orlandini von Niessen, A. G. et al., Mol Ther 27, 824-836;2019; Holtkamp, S. et al., Blood 108, 4009-4017, 2006). Non-codingbackbone elements included the regions from the T7 promoter to the 5′and 3′ UTR plus a poly(A) tail (100 nucleotides) interrupted by a linker(A30LA70, 10 nucleotides). The DNA was purified, spectrophotometricallyquantified, and in vitro transcribed by T7 RNA polymerase in thepresence of a trinucleotide cap1 analogue ((m₂^(7,3′-O))Gppp(m^(2′-O))ApG; TriLink) and ofN¹-methylpseudouridine-5′-triphosphate (m1ψTP; Thermo Fisher Scientific)instead of uridine-5′-triphosphate (UTP) (Grudzien-Nogalska, E. et al.,Methods in molecular biology (Clifton, N.J.) 969, 55-72, 2013). RNA waspurified using magnetic particles (Berensmeier, S., Appl. Microbiol.Biotechnol. 73, 495-504, 2006), integrity assessed by microfluidiccapillary electrophoresis (Agilent Fragment Analyser), andconcentration, pH, osmolality, endotoxin level and bioburden determined.

Lipid-Nanoparticle Formulation of the RNA.

Purified RNA was formulated into LNPs using an ethanolic lipid mixtureof ionisable cationic lipid and transferred into an aqueous buffersystem via diafiltration to yield an LNP composition similar to onepreviously described (Maier, M. A. et al., Molecular therapy: thejournal of the American Society of Gene Therapy 21, 1570-1578, 2013).BNT162b1 was stored at −70° C. at a concentration of 0.5 mg/mL.

mRNA Transfection.

HEK293T/17 cells were transfected with transfection reagent-mixedBNT162b1 RNA or BNT162b1 by incubation for 18 hours. Non-LNP formulatedmRNA (1 μg for Western blot and flow cytometry, 2.5 μg forimmunofluorescence) was diluted in Opti-MEM medium (Thermo FisherScientific) and mixed with the transfection reagents according to themanufacturer's instructions (RiboJuice, Merck Millipore).

Western Blot Analysis.

A lysate of BNT162b1 RNA transfected HEK293T/17 cells was analysed bydenaturing SDS-PAGE with 10% Mini-Protean TGX precast polyacrylamidegels (Bio-Rad) and Western blot. Transfer to a nitrocellulose membrane(Carl Roth) was performed using a semi-dry transfer system (Trans-BlotTurbo Transfer System, Bio-Rad). Blotted proteins were detected with aprimary rabbit polyclonal antibody elicited by a recombinant S1 fragmentof the SARS-CoV S (SinoBiological) and a secondary anti-rabbit horseraddish peroxidase (HRP)-conjugated antibody (Sigma Aldrich). Blots weredeveloped with SuperSignal West Femto chemiluminescent substrate (ThermoFisher Scientific) and imaged with a Bio-Rad ChemiDoc system using theImage Lab software version 5.0.

Immunofluorescence.

Transfected HEK293T/17 cells were fixed in 4% paraformaledehyde (PFA)and permeabilised in phosphate-buffered saline (PBS)/0.2% Triton X-100.Free binding sites were blocked and cells incubated with a rabbitpolyclonal antibody recognising the S1 Subunit (SinoBiological) andanti-rabbit IgG secondary antibody (Jackson ImmunoResearch), or labelledConcanavalin A (Invitrogen). DNA was stained with Hoechst (LifeTechnologies). Images were acquired with a Leica SP8 confocalmicroscope.

Flow Cytometry.

Transfected HEK293T/17 cells were stained with Fixable Viability Dye(eBioscience). After fixation (Fixation Buffer, Biolegend), cells werepermeabilised (Perm Buffer, eBioscience) and stained with a monoclonalSARS-CoV-2 spike S1 antibody (SinoBiological). Cells were acquired on aFACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva softwareversion 8.0.1 and analysed by FlowJo software version 10.6.2 (FlowJoLLC, BD Biosciences).

For mouse T-cell analysis in peripheral blood, erythrocytes from 50 μLfreshly drawn blood were lysed (ACK lysing buffer, Gibco), and cellswere stained with Fixable Viability Dye (eBioscience) and primaryantibodies in the presence of Fc block in flow buffer (DPBS [Gibco]supplemented with 2% FCS, 2 mM EDTA [both Sigma] and 0.01% sodium azide[Morphisto]). After staining with secondary biotin-coupled antibodies inflow buffer, cells were stained extracellularly against surface markerswith directly labelled antibodies and streptavidin in Brilliant StainBuffer Plus (BD Bioscience) diluted in flow buffer. Cells were fixedwith 2% RotiHistofix (Carl Roth) and permeabilised (Perm Buffer,FoxP3/Transcription Factor Staining Buffer Set, eBioscience) overnight.Permeabilised cells were intracellularly treated with Fc block andstained with antibodies against transcription factors in Perm Buffer.

For mouse T-cell analysis in lymphoid tissues, 1×10⁶ lymph node and4×10⁶ spleen cells were stained for viability and extracellular antigenswith directly labelled antibodies. Cells were washed in 2% RotiHistofixand fixed (Fix/Perm Buffer, FoxP3/Transcription Factor Staining BufferSet, eBioscience) overnight. Intracellular staining was performed asdescribed for blood T-cell staining. For mouse B-cell subtyping inlymphoid tissues, 2.5×10⁵ lymph node and 1×10⁶ spleen cells were treatedwith Fc block, stained for viability and extracellular antigens asdescribed for blood T-cell staining and fixed with 2% RotiHistofixovernight. For mouse intracellular cytokine staining in T cells, 1×10⁶lymph node and 4×10⁶ spleen cells were ex vivo restimulated with 0.2μg/mL final concentration per peptide of full-length S peptide mix inthe presence of GolgiStop and GolgiPlug (both BD Bioscience) for 5hours. Cells were stained for viability and extracellular antigens asdescribed for lymphoid T-cell staining. Cells were fixed with 2%RotiHistofix and permeabilised overnight. Intracellular staining wasperformed as described for blood T-cell staining.

Mouse cells were acquired on a BD Symphony A3 or BD Celesta (B-cellsubtyping) flow cytometer (BD Bioscience) using BD FACSDiva softwareversion 9.1 or 8.0.1.1, respectively, and analysed with FlowJo 10.6(FlowJo LLC, BD Biosciences).

Protein Expression and Purification.

To express the RBD-foldon encoded by BNT162b1 for biochemical andstructural analysis, DNA corresponding to the RNA coding sequence wascloned into the pMCG1309 vector. A plasmid encoding amino acids 1-615 ofhuman ACE2 with C-terminal His-10 and Avi tags was generated fortransient expression of the ACE2 peptidase domain (ACE2 PD) in Expi293Fcells. The ACE2/B⁰AT1 complex was produced by co-expression of twoplasmids in Expi293F cells, one of them encoding ACE2 amino acids 1-17followed by haemagglutinin and Strep II tags and ACE2 amino acids18-805, and the other containing a methionine followed by a FLAG tag andamino acids 2-634 of human B⁰AT1.

Secreted ACE2 PD was isolated from conditioned cell culture medium usingNickel Excel resin (GE Healthcare) followed by gel filtrationchromatography on a Superdex200 10/30 column (GE Healthcare) in PBS.Approximately 5 mg of purified ACE2 PD was covalently attached per 1 mLof 4% beaded agarose by amine coupling using AminoLink Plus resin(Thermo Fisher Scientific). The RBD-trimer was purified from conditionedmedium by affinity capture with the ACE2 PD crosslinked agarose and waseluted from the resin with 3 M MgCl₂. Following dialysis, the proteinwas concentrated and purified by gel filtration using a Superdex20010/300 column in HEPES-buffered saline (HBS) with 10% glycerol.Purification of the ACE2/B⁰AT1 complex was based on the proceduredescribed previously (Yan, R. et al., Science (New York, N.Y.) 367,1444-1448, 2020). To form the ACE2/B⁰AT1/RBD-trimer complex, ACE2/B⁰AT1aliquots were combined with purified RBD-foldon diluted in ACE2/B⁰AT1size exclusion chromatography buffer (25 mM Tris pH 8.0, 150 mM NaCl,0.02% glyco diosgenin) for a 3:1 molar ratio of RBD-trimers to ACE2protomers. After incubation at 4° C. for 30 minutes, the sample wasconcentrated and resolved on a Superose 6 Increase 10/300 GL column.Peak fractions containing the complex were pooled and concentrated.

Surface Plasmon Resonance Spectroscopy.

Binding kinetics of murine RBD-specific serum IgGs was determined usinga Biacore T200 device (Cytiva) with HBS-EP running buffer (BR100669,Cytiva) at 25° C. Carboxyl groups on the CM5 sensor chip matrix wereactivated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) to formactive esters for the reaction with amine groups. Anti-mouse-Fc-antibody(Jackson ImmunoResearch) was diluted in 10 mM sodium acetate buffer pH 5(30 μg/mL) for covalent coupling to immobilisation level of ^(˜)10,000response units (RU). Free N-hydroxysuccinimide esters on the sensorsurface were deactivated with ethanolamine.

Mouse serum was diluted 1:50 in HBS-EP buffer and applied at 10 μL/minfor 30 seconds to the active flow cell for capture by immobilisedantibody, while the reference flow cell was treated with buffer. Bindinganalysis of captured murine IgG antibodies to RBD-His (Sino BiologicalInc.) was performed using a multi-cycle kinetic method withconcentrations ranging from 1.5625 to 50 nM. An association period of180 seconds was followed by a dissociation period of 600 seconds with aconstant flow rate of 40 μL/min and a final regeneration step. Bindingkinetics were calculated using a global kinetic fit model (1:1 Langmuir,Biacore T200 Evaluation Software Version 3.1, Cytiva).

Biolayer Interferometry.

Binding of RBD-foldon to the ACE2-PD was measured by biolayerinterferometry on an Octet RED384 (FortéBio) in a buffer composed of 10mM HEPES pH 7.5, 150 mM NaCl and 1 mM EDTA at 25° C. Avi-tagged humanACE2 PD was immobilised on streptavidin-coated sensors. Binding datawere collected for 10 minutes of association and 15 minutes ofdissociation for a concentration series of RBD-trimer. Data werereference-subtracted and fit to a 1:1 binding model with R² valuegreater than 0.96 to determine kinetics and affinity of binding, usingOctet Data Analysis Software v10.0 (FortéBio). The dissociation rate ofinteraction (k_(d)) was slower than the limit of measurement of theinstrument, and the binding affinity (K_(D)) was estimated using anassumed dissociation rate k_(d) of 1×10⁻⁶ s⁻¹.

Electron Microscopy of Negatively Stained Samples.

Purified RBD-trimer protein in 4 μL was applied to a glow-dischargedcopper grid overlaid with formvar and amorphous carbon (Ted Pella).Staining was performed with Nano-W organotungstate stain (Nanoprobes)according to the manufacturer's protocol, and the sample imaged using anFEI TF-20 microscope operating at 200 kV, with a magnification of62,000× and defocus of −2.5 μm. Micrographs were contrast transferfunction (CTF)-corrected in RELION using CTFFIND-4.1 (Rohou, A. &Grigorieff, N., Journal of structural biology 192, 216-221, 2015). Asmall manually picked dataset was used to generate 2D references forauto-picking. The resulting particle set was subjected to 2Dclassification in RELION 3.0.6 (Zivanov, J. et al., eLife 7;10.7554/eLife.42166 (2018)).

Cryo-Electron Microscopy.

Purified ACE2/B⁰AT1/RBD-trimer complex at 6 mg/mL in 4 μL was applied togold Quantifoil R1.2/1.3 200 mesh grids glow discharged in residual airfor 30 seconds at 20 mA using a Pelco Easiglow. The sample was blottedusing a Vitrobot Mark IV for 5 seconds with a force of −3 before beingplunged into liquid ethane cooled by liquid nitrogen. In total, 7,455micrographs were collected from a single grid on a Titan Krios operatingat 300 keV equipped with a Gatan K2 Summit direct electron detector insuper-resolution mode at a magnification of 165,000×, for a magnifiedpixel size of 0.435 Å at the specimen level. Data were collected over adefocus range of −1.2 to −3.4 μm with a total electron dose of 52.06e⁻/Å² fractionated into 40 frames over a 6-second exposure for 1.30e⁻/Å²/frame. Initial motion correction was performed in Warp (Tegunov,D. & Cramer, P., Nature methods 16, 1146-1152, 2019), during whichsuper-resolution data were binned to give a pixel size of 0.87 Å.Corrected micrographs were imported into RELION 3.1-beta (Zivanov, J. etal., eLife 7; 10.7554/eLife.42166 (2018)) for CTF estimation withCTFFIND-4.1 (Rohou, A. & Grigorieff, N., Journal of structural biology192, 216-221, 2015). Particles were picked using theLaPlacian-of-Gaussian particle picking algorithm as implemented inRELION and extracted with a box size of 450 pixels. References obtainedby 2D classification were used for a second round of reference-basedauto-picking, yielding a dataset of 715,356 particles. Particleheterogeneity was filtered out with 2D and 3D classification with a masksize of 280 nm to filter out the non-ACE2-bound RBD copies in eachRBD-trimer, yielding a set of 87,487 particles, which refined to 3.73 Åwith C2 symmetry. Refinement after subtraction of micelle and B⁰AT1density from the particles yielded an improved map of 3.24 Å. The atomicmodel from PDB ID 6M17 (Yan, R. et al., Science (New York, N.Y.) 367,1444-1448, 2020) was rigid-body fitted into the 3.24 Å density, thenflexibly fitted to the density using real-space refinement in Phenix(Adams, P. D. et al., Acta crystallographica. Section D, Biologicalcrystallography 66, 213-221, 2010) alternating with manual building inCoot (Emsley, P. et al., Acta crystallographica. Section D, Biologicalcrystallography 66, 486-501, 2010). The microscope was operated forimage acquisition using SerialEM software version 3.8.0 beta(Mastronarde, D. N., Journal of structural biology 152, 36-51, 2005).Biolayer interferometry data was collected with Octet Data Acquisitionsoftware version 10.0.0.87 and processing was performed using ForteBioData Analysis software version 10.0.

Immunisation.

Mice. Female BALB/c mice (Janvier; 8-12 weeks) were randomly allocatedto groups. BNT162b1 was diluted in PBS, 300 mM sucrose or saline (0.9%NaCl) and injected IM into the gastrocnemius muscle at a volume of 20 μLunder isoflurane anaesthesia.

Rhesus macaques (Macaca mulatta). Male Rhesus macaques (2-4 years) wererandomly assigned to receive either BNT162b1 or saline placebo controlin 0.5 mL volume administered by IM injection in the left quadricepsmuscle on Days 0 and 21. Blood for serum and PBMCs was collected incompliance with animal protocol 2017-8725-023 approved by the NIRCInstitutional Animal Care and Use Committee. Animals were anesthetisedwith ketamine HCl (10 mg/kg; IM) during blood collection andimmunisation, and monitored for adequate sedation.

SARS-CoV-2 Challenge of Rhesus Macaques.

The SARS-CoV-2 inoculum was obtained from a stock of 2.1×10⁶ PFU/mLpreviously prepared at Texas Biomedical Research Institute (San Antonio,Tex.), aliquoted into single use vials, and stored at −70° C. Theworking virus stock was generated from two passages of the SARS-CoV-2USA-WA1/2020 isolate (a 4^(th) passage seed stock purchased from BEIResources; NR-52281) in Vero E6 cells. The virus was confirmed to beSARS-CoV-2 by deep sequencing and identical to the published sequence(GenBank accession number MN985325.1). BNT162b1-immunised (n=6) andage-matched saline control-immunised (n=6) male Rhesus macaques(control) were challenged with 1×10⁶ plaque forming units of SARS-CoV-2USA-WA1/2020 isolate, split equally between the intranasal (IN; 0.2 mL)and intratracheal (IT; 0.2 mL) routes as previously described (Singh, D.K. et al., SARS-CoV-2 infection leads to acute infection with dynamiccellular and inflammatory flux in the lung that varies across nonhumanprimate species, 2020). The challenge was performed 41 to 48 days afterthe second immunisation. A separate sentinel group of non-immunised age-and sex-matched animals (n=3) received only DMEM supplemented with 10%FCS IN (0.2 mL) and IT (0.2 mL). Approximately two weeks prior tochallenge, animals were moved to the Animal Biosafety Level 3 (ABSL-3)facility at Southwest National Primate Research Center (SNPRC; SanAntonio, Tex.). Animals were monitored regularly by a board-certifiedveterinary clinician for rectal body temperature, weight and physicalexamination. Specimen collection was performed under tiletaminezolazepam (Telazol) anaesthesia as described (Singh, D. K. et al.,SARS-CoV-2 infection leads to acute infection with dynamic cellular andinflammatory flux in the lung that varies across nonhuman primatespecies, 2020). Nasal swabs were collected from macaques at 0, 1, 3, and6 days after inoculation to assess viral titers. Bronchoalveolar lavage(BAL) was performed the week before challenge and at Days 3 and 6post-inoculation by instilling four times 20 mL of saline. Thesewashings were pooled, aliquoted and stored frozen at −70° C.

Reverse-Transcription Quantitative Polymerase Chain Reaction.

To detect and quantify SARS-CoV-2, viral RNA was extracted from nasalswabs and BAL specimens as previously described (Mehra, S. et al., TheJournal of infectious diseases 207, 1115-1127, 2013; Gautam, U. S. etal., Proceedings of the National Academy of Sciences of the UnitedStates of America 115, E62-E71; 2018; Joosten, S. A. et al., PLoSpathogens 6, e1000782, 2010) and tested by RT-qPCR as previouslydescribed (Singh, D. K. et al., SARS-CoV-2 infection leads to acuteinfection with dynamic cellular and inflammatory flux in the lung thatvaries across nonhuman primate species, 2020). Briefly, 10 μg yeast tRNAand 1×10³ PFU of MS2 phage (Escherichia coli bacteriophage MS2, ATCC)were added to each thawed sample, and RNA extraction performed using theNucleoMag Pathogen kit (Macherey-Nagel). The SARS-CoV-2 RT-qPCR wasperformed on extracted RNA using a CDC-developed 2019-nCoV_N1 assay on aQuantStudio 3 instrument (Applied Biosystems). The cut-off forpositivity (limit of detection, LOD) was established at 10 geneequivalents (GE) per reaction (800 GE/mL). Samples were tested induplicate. On day 6, one BAL specimen from the control group and one day1 nasal swab from the BNT162b1-immunised group had, on repeatedmeasurements, viral RNA levels on either side of the LLOD. Thesespecimens were categorised as indeterminate and excluded from the graphsand the analysis.

Tissue Preparation.

Mice. Peripheral blood was collected from the retro-orbital venousplexus or vena facialis under isoflurane anaesthesia. Blood wascentrifuged for 5 minutes at 16.000×g, and the serum was immediatelyused for downstream assays or stored at −20° C. Spleen single-cellsuspensions were prepared in PBS by mashing tissue against the surfaceof a 70 μm cell strainer (BD Falcon) using the plunger of a 3-mL syringe(BD Biosciences). Erythrocytes were removed by hypotonic lysis.Popliteal, inguinal and iliac lymph nodes were pooled, cut into pieces,digested with collagenase D (1 mg/mL; Roche) and passed through cellstrainers. Rhesus macaques (Macaca mulatta). Blood for serum and PBMCswas collected in compliance with animal protocol 2017-8725-023 approvedby the NIRC Institutional Animal Care and Use Committee.

RBD-Binding IgG Antibody Assay.

For mouse sera, MaxiSorp plates (Thermo Fisher Scientific) were coatedwith recombinant RBD (100 ng/100 μL) in sodium carbonate buffer, andbound IgG was detected using an HRP-conjugated secondary antibody andTMB substrate (Biotrend). Data collection was performed using a BioTekEpoch reader and Gen5 software version 3.0.9. For concentrationanalysis, the signal of the specific samples was correlated to astandard curve of an isotype control. For Rhesus macaque and human sera,a recombinant SARS-CoV-2 RBD containing a C-terminal Avitag™ (AcroBiosystems) was bound to streptavidin-coated Luminex microspheres. BoundRhesus macaque or human anti-RBD antibodies present in the serum weredetected with a fluorescently labelled goat anti-human polyclonalsecondary antibody (Jackson ImmunoResearch). Data were captured asmedian fluorescent intensities (MFIs) using a Bioplex200 system(Bio-Rad) and converted to U/mL antibody concentrations using areference standard curve consisting of 5 pooled human COVID-19convalescent serum samples (obtained >14 days PCR diagnosis), diluted inantibody depleted human serum, with arbitrary assigned concentrations of100 U/mL and accounting for the serum dilution factor.

VSV-SARS-CoV-2 Spike Variant Pseudovirus Neutralisation.

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes GFP instead of VSV-G (VSVΔG-GFP) was pseudotypedwith SARS-CoV-2 S protein according to published pseudotyping protocols(Berger Rentsch, M. & Zimmer, G., PLoS ONE 6, e25858, 2011; Lester, S.et al., Access Microbiology 1, 20290, 2019). In brief, HEK293T/17monolayers transfected to express SARS-CoV-2 S truncated of theC-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S-CΔ19) wereinoculated with VSVΔG-GFP vector. After incubation for 1 hour at 37° C.,the inoculum was removed and cells were washed with PBS before mediumsupplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) wasadded to neutralise residual input virus. VSV/SARS-CoV-2pseudovirus-containing medium was harvested 20 hours after inoculation,0.2 μm filtered and stored at −80° C.

Vero-76 cells were seeded in 96-well plates. Serial dilutions of mouseserum samples were prepared and pre-incubated for 10 minutes at roomtemperature with VSV/SARS-CoV-2 pseudovirus suspension (4.8×10³infectious units [IU]/mL) before transferring the mix to Vero-76 cells.Inoculated Vero-76 cells were incubated for 20 hours at 37° C. Plateswere placed in an IncuCyte Live Cell Analysis system (Sartorius) andincubated for 30 minutes prior to the analysis (IncuCyte 2019B Rev2software). Whole well scanning for brightfield and GFP fluorescence wasperformed using a 4× objective. The 50% pseudovirus neutralisation titre(pVNT₅₀) was reported as the reciprocal of the first serum dilutionyielding a 50% reduction in GFP-positive infected cell number per wellcompared to the mean of the no serum pseudovirus positive control. Eachserum sample dilution was tested in duplicates.

SARS-CoV-2 Neutralisation by Human Convalescent and Rhesus Macaque Sera.

The SARS-CoV-2 neutralisation assay used a previously described strainof SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse geneticsand engineered by the insertion of an mNeonGreen (mNG) gene into openreading frame 7 of the viral genome (Xie, X. et al., Cell host & microbe27, 841-848.e3, 2020). This reporter virus generates similar plaquemorphologies and indistinguishable growth curves from wild-type virus.Viral master stocks were grown in Vero E6 cells as previously described(Lester, S. et al., Access Microbiology 1, 20290, 2019). When testinghuman convalescent serum specimens, the fluorescent neutralisation assayproduced comparable results as the conventional plaque reductionneutralisation assay. Serial dilutions of heat-inactivated sera wereincubated with the reporter virus (2×10⁴ PFU per well) to yieldapproximately a 10-30% infection rate of the Vero CCL81 monolayer) for 1hour at 37° C. before inoculating Vero CCL81 cell monolayers (targetedto have 8,000 to 15,000 cells per well) in 96-well plates to allowaccurate quantification of infected cells. Total cell counts per wellwere enumerated by nuclear stain (Hoechst 33342) and fluorescent virallyinfected foci were detected 16-24 hours after inoculation with aCytation 7 Cell Imaging Multi-Mode Reader (Biotek) with Gen5 Image Primeversion 3.09. Titers were calculated in GraphPad Prism version 8.4.2 bygenerating a 4-parameter (4PL) logistical fit of the percentneutralisation at each serial serum dilution. The 50% neutralisationtitre (VNT₅₀) was reported as the interpolated reciprocal of thedilution yielding a 50% reduction in fluorescent viral foci.

IFNγ ELISpot.

ELISpot assays were performed with mouse IFNγ ELISpot^(PLUS) kitsaccording to the manufacturer's instructions (Mabtech). A total of 5×10⁵splenocytes was ex vivo were restimulated with the full-length S peptidemix (0.1 μg/mL final concentration per peptide, JPT) or controls(gp70-AH1 [SPSYVYHQF (SEQ ID NO: 35)] (Slansky, J. E. et al., Immunity13, 529-538, 2000), JPT, 4 μg/mL; Concanavalin A (ConA), Sigma, 2μg/mL). Streptavidin-ALP and BCIP/NBT-plus substrate were added, andspots counted using an ELISpot plate reader (ImmunoSpot® S6 CoreAnalyzer, CTL). Spot numbers were evaluated using ImmunoCapture ImageAquision Software V7.0 and ImmunoSpot 7.0.17.0 Professional. For T-cellsubtyping, CD8⁺ T cells were isolated from splenocyte suspensions usingMACS MicroBeads (CD8a [Ly-2], Miltenyi Biotec) according to themanufacturer's instructions. The flow-through served as a source of CD4⁺T cells. CD8⁺ or CD4⁺ T cells were subsequently restimulated withsyngeneic bone marrow-derived dendritic cells loaded with full-length Speptide mix (0.1 μg/mL final concentration per peptide) or medium ascontrol. Purity of isolated T-cell subsets was determined by flowcytometry to calculate spot counts per 1×10⁵ CD8⁺ or CD4⁺ T cells.

Cytokine Profiling.

Mouse splenocytes were re-stimulated for 48 hours with full-length Speptide mix (0.2 μg/mL final concentration per peptide) or medium only.Concentrations of IFNγ, IL-2, IL-4 and IL-5 in supernatants weredetermined using a bead-based, 11-plex T_(H)1/T_(H)2 mouse ProcartaPlexmultiplex immunoassay (Thermo Fisher Scientific) according to themanufacturer's instructions. Fluorescence was measured with a Bioplex200system (Bio-Rad) and analysed with ProcartaPlex Analyst 1.0 software(Thermo Fisher Scientific).

Statistics and Reproducibility.

No statistical methods were used to predetermine group and samples sizes(n). All experiments were performed once. P-values reported for RT-qPCRanalysis were determined by categorical analysis for binomial response(undetectable viral RNA after challenge as success, measurable viral RNAafter challenge as failure) with logit link to treatment and day effectsusing PROC GENMOD from SAS® 9.4. Samples from post challenge days (Days3 and 6 for BAL; Days 1, 3 and 6 for nasal swab) were included in theanalysis. Indeterminate results were excluded from this analysis. Allremaining analyses were carried out using GraphPad Prism 8.4.

Results

We designed a SARS-CoV-2 vaccine named BNT162b1, which is composed of anLNP-encapsulated N¹-methyl-pseudouridine (m1ψ) nucleoside-modified mRNAthat encodes the RBD fused at its C-terminus to the naturaltrimerisation domain (foldon) of T4 fibritin (Meier, S. et al., Journalof molecular biology 344, 1051-1069, 2004) (FIG. 64 a ). The SARS-CoV-2S signal peptide (SP) enables ER translocation and secretion of thetrimeric RBD. The m1W-modification of the RNA dampens innate immunesensing and, together with optimized non-coding sequence elements,increases RNA translation in vivo (Orlandini von Niessen, A. G. et al.,Mol Ther 27, 824-836, 2019; Karikó, K. et al., Molecular therapy: thejournal of the American Society of Gene Therapy 16, 1833-1840, 2008).

BNT162b1 RNA in vitro transcribed by T7 polymerase from a plasmid DNAtemplate had a single, sharp peak microfluidic capillary electrophoresisprofile, consistent with its calculated length of 1262 nucleotides,indicating purity and integrity (data not shown). Western blot analysisof a lysate of BNT162b1 RNA-transfected HEK293T/17 cells demonstratedthat the RBD was expressed from the RNA and had an apparent molecularweight consistent with its calculated weight of 29.46 kDa (data notshown). Protein expression and endoplasmic reticulum localisation on thesecretory pathway in transfected cells were confirmed by flow cytometryand immunofluorescence microscopy, respectively (data not shown).

For structural characterization, the trimerised RBD was expressed from aDNA sequence corresponding to the coding sequence of BNT162b1 RNA inExpi293F cells and purified by affinity capture with the ACE2 peptidasedomain immobilized on agarose beads. The trimerised RBD bound to thehuman ACE2 peptidase domain (PD) with high affinity (5 pM K_(D)), whichis approximately 1,000-fold the reported K_(D) of 5.09 nM for monomericRBD and consistent with the avidity effect of multimeric binding (datanot shown). The trimeric valency of the RBD-foldon and its flexibilitywere visualized by electron microscopy (EM) of negatively stainedspecimens, which revealed a range of conformations (FIG. 64 b ).Although the flexibility of the RBD-foldon precluded direct structuralanalysis at high resolution, the RBD domains could be immobilized bybinding to a complex of ACE2 and the B⁰AT1 neutral amino acidtransporter, which ACE2 chaperones, when that complex was in thepreviously reported closed conformation (Yan, R. et al., Science (NewYork, N.Y.) 367, 1444-1448, 2020). The size and symmetry of theRBD-foldon/ACE2/B⁰AT1 ternary complex aided image reconstruction byelectron cryomicroscopy (cryoEM), and the structure of the RBD domainsin the complex was determined to 3.24 Å resolution (FIG. 64 c ). Onecopy of the RBD was resolved for each bound trimer. The bindinginterface between the resolved RBD and the ACE2 extracellular domain wasfitted to a previously reported structure and showed good agreement (He,Y. et al., Biochemical and Biophysical Research Communications 324,773-781, 2004; Yi, C. et al., Cellular & molecular immunology;10.1038/s41423-020-0458-z, 2020). The high affinity binding to ACE2 andwell-resolved structure in complex with ACE2 demonstrate that therecombinant RBD-foldon authentically presents the ACE2 binding sitetargeted by many SARS-CoV-2 neutralising antibodies (Brouwer, P. J. M.et al., Science (New York, N.Y.); 10.1126/science.abc5902 (2020); Zost,S. J. et al., Nature medicine; 10.1038/s41591-020-0998-x (2020)).

BNT162b1-elicited B- and T-cell immune responses were characterised in aseries of experiments in BALB/c mice after a single intramuscular (IM)immunisation with 0.2, 1, or 5 μg BNT162b1 or with buffer alone.RBD-specific serum IgG developed quickly at all dose levels in adose-dependent manner and plateaued around day 21 (at 1.63±0.13 mg/mLfor the 5 μg dose level; FIG. 65 a ). Vaccine-elicited IgG had highRBD-binding affinity (geometric mean K_(D) 48.0 pM) with high on-rate(geometric mean k_(on) 1.72×10⁶/Ms) and low off-rate (geometric meanK_(off) 8.27×10⁻⁵/s; FIG. 65 b ). SARS-CoV-2 neutralising activity inmouse serum was measured by a vesicular stomatitis virus (VSV)-basedSARS-CoV-2 pseudovirus neutralisation assay. Mean 50% pseudovirusneutralisation titers (pVNT₅₀) increased steadily after immunisation to102, 192, and 1,056 on day 28 for the 0.2, 1, and 5 μg dose levels,respectively (FIG. 65 c ).

A high fraction of splenocytes of both CD4⁺ and CD8⁺ T-cell phenotypesisolated from BNT162b1-immunised mice on days 12 and 28 afterimmunisation, when re-stimulated ex vivo with a full-length S peptidemix, exerted a strong antigen-specific IFNγ-response in ELISpot assays(FIG. 65 d ). Full-length S peptide-stimulated bulk splenocytes and CD4⁺and CD8⁺ subsets also show high IFNγ production at day 12 andsignificant IL-2 responses but much lower IL-4 responses in flowcytometric cytokine release analyses, indicating a T_(H)1 phenotyperesponse (FIG. 65 e ). The T_(H)1 phenotype persists, with totalsplenocytes harvested on day 28 and stimulated with the full-length Speptide pool producing high levels of IL-2 and IFNγ but undetectableamounts of the T_(H)2 cytokines IL-4 and IL-5 in multiplex immunoassays(FIG. 65 f ). In draining lymph nodes (dLN) and spleens obtained 12 daysafter immunisation of mice with BNT162b1 or buffer, much higher numbersof B cells (including plasma cells, class switched IgG1- andIgG2a-positive B cells, and germinal center B cells) were observed inthe samples from mice that received BNT162b1 (data not shown). In bloodobtained 7 days after immunisation, the number of circulating B cellswas lower than in buffer-immunised mice, most likely due to B-cellhoming to lymphoid compartments (data not shown). dLNs fromBNT162b1-immunised mice also displayed an elevation in T-cell counts,particularly numbers of T follicular helper (T_(FH)) cells, includingsubsets with ICOS upregulation, which are known to play an essentialrole in the formation of germinal centers (Hutloff, A., Oncotarget 6,21785-21786, 2015) (data not shown). BNT162b1-induced elevation ofT_(FH) cells was also detected in the spleen and blood (data not shown).In aggregate, these data indicate a strong and concurrent induction ofSARS-CoV-2 S-specific neutralising antibody titers and a T_(H)1-drivenT-cell response by BNT162b1. Intramuscularly administered BNT162b1appears to be delivered to dLNs as immune-educated sites for proficientvaccine priming, with migration of lymphocytes from the blood tolymphoid tissues to participate in the vaccine response.

The immunogenicity of BNT162b1 was next tested in 2-4 year old maleRhesus macaques. Groups of six were immunised IM with 30 or 100 μg ofBNT162b1 or with saline control on Days 0 and 21. RBD-binding IgG wasreadily detectable by Day 14 after a single immunisation, and levelsincreased further through Day 21, when the boosting dose was given (FIG.66 a ). Seven days after the second immunisation (Day 28), the geometricmean RBD-binding IgG concentrations (GMCs) were 20,962 units (U)/mL (30μg dose level) and 48,575 U/mL (100 μg dose level). For comparison, theRBD-binding IgG GMC of a panel of 38 SARS-CoV-2 convalescent human serawas 602 U/mL, substantially lower than the GMC of the immunised Rhesusmacaques after one or two doses. Fifty percent neutralisation titers(VNT₅₀), measured by an authentic SARS-CoV-2 neutralisation assay(Muruato, A. E. et al., bioRxiv: the preprint server for biology;10.1101/2020.05.21.109546, 2020), were detectable in Rhesus sera by Day14 after a single immunisation and reached geometric mean titers (GMTs)of 768 (30 μg dose level) or 1,714 (100 μg dose level) 7 days after theboost (Day 28, FIG. 66 b ). Robust neutralisation GMTs of 247 for 30 μgand 564 for 100 μg dose levels persisted to at least Day 42 (most recenttime point tested). For comparison, the 50% neutralisation GMT of thehuman convalescent serum panel was 93.6.

The groups of Rhesus macaques (n=6) that had received two immunisationswith 100 μg BNT162b1 or buffer control were challenged 41 to 48 daysafter the second immunisation with 1×10⁶ plaque forming units ofSARS-CoV-2 (strain USA-WA1/2020), split equally between the intranasaland intratracheal routes, as previously described (Singh, D. K. et al.SARS-CoV-2 infection leads to acute infection with dynamic cellular andinflammatory flux in the lung that varies across nonhuman primatespecies, 2020). Three non-immunised, age-matched, male Rhesus macaques(sentinel) were mock-challenged with cell culture medium. At the time ofchallenge, SARS-CoV-2 neutralising titers ranged from 208 to 1,185 inthe BNT162b1-immunised animals and were undetectable in animals from thecontrol-immunised and sentinel groups.

SARS-CoV-2 RNA was measured in bronchoalveolar lavage (BAL) and nasalswab samples by reverse-transcription quantitative polymerase chainreaction (RT-qPCR). All BAL and nasal swab samples obtained before theinfectious challenge and all those obtained from sentinel animals lackeddetectable SARS-CoV-2 RNA (FIG. 67 ). Three days after SARS-CoV-2challenge, viral RNA was detected in BAL fluid from 5/6control-immunised and 2/6 BNT162b1-immunised animals (FIG. 67 a ). By 6days after challenge, all six BNT162b1-immunised macaques hadundetectable viral RNA in their lungs; of the control-immunisedmacaques, three had a high level of viral RNA in BAL fluid, two hadcleared, and one had an indeterminate RT-qPCR result. At the time ofnecropsy (7-23 days after challenge), no viral RNA was detectable in BALfluid from any animal. After SARS-CoV-2 challenge, viral RNA wasdetected in nasal swabs of the control-immunised group at each timepoint: two animals at Day 1, three animals at Days 3 and 6 afterchallenge (FIG. 67 b ), and two animals at the time of necropsy (notshown). In BNT162b1-immunised animals, all nasal swabs were negative orindeterminate at Day 1, and all were negative at Day 3 and at the timeof necropsy; at Day 6, swabs from two were positive, indicating a moretransient course of viral RNA detection compared to non-immunised Rhesusmacaques. The difference in the proportion of animals with detectableviral RNA between BNT162b1-immunised animals and control-immunisedanimals is statistically significant (p=0.0037 for BAL, and 0.0212 fornasal swab). None of the challenged animals showed clinical orradiographic signs of significant illness, indicating that the 2-4 yearsold male Rhesus challenge model is primarily an infection model forSARS-CoV-2, not a COVID-19 disease model.

Discussion

We demonstrate that BNT162b1, an LNP-formulated m1W nucleoside-modifiedmRNA encoding the trimeric RBD antigen, is highly immunogenic in miceand Rhesus macaques and limits infection in Rhesus macaques challengedwith infectious SARS-CoV-2. The RBD-foldon coding sequence directs theexpression of a flexible, trimeric protein that binds ACE2 with highaffinity and has a structurally intact ACE2 receptor binding site. Onekey finding is that in mice, a single sub-microgram immunisation rapidlyinduces high neutralising antibody titers that are in the range or aboverecently reported SARS-CoV-2 vaccine candidates (van Doremalen, N. etal., bioRxiv: the preprint server for biology; 10.1101/2020.05.13.093195(2020); Corbett, K. S. et al., bioRxiv: the preprint server for biology;10.1101/2020.06.11.145920 (2020)). The strong CD4⁺ and stronger CD8⁺T-cell responses, both skewing towards a T_(H)1-bias, and T_(FH)generation may imply a strong protection capacity induced by the vaccinecandidate (Pardi, N. et al., The Journal of Experimental Medicine 215,1571-1588, 2018). Proliferation of T_(FH) in germinal centres isintegral for generation of an adaptive B-cell response. In humans,T_(FH) occurring in the circulation after vaccination were correlatedwith a high frequency of antigen-specific antibodies (Farooq, F. et al.,Scientific reports 6, 27944, 2016). Immunisation with BNT162b1 triggeredredistribution of B cells and T_(FH) cells from the blood to lymphoidtissues, where antigen presentation occurs.

Another significant finding is that in Rhesus macaques two doses of m1Wnucleoside-modified mRNA encoding the trimeric SARS-CoV-2 S RBD-foldonelicited SARS-CoV-2 neutralising GMTs 8.2 to 18.2-fold the GMT of aSARS-CoV-2 convalescent human serum panel. Results in nonhuman primatesconfirm the vaccine's high potency and ability to protect againstSARS-CoV-2 challenge in a preclinical model of acute SARS-CoV-2infection.

Example 11: A RNA Vaccine Encoding the Prefusion-Stable SARS-CoV-2 S isHighly Immunogenic in Mice and Non Human Primates

Here, we report a SARS-CoV-2 infectious challenge of immunised macaqueswith BNT162b2 vaccine.

Materials and Methods Manufacturing of In Vitro Transcribed RNA.

To generate the template for RNA synthesis, a DNA fragment encoding thefull-length SARS-CoV-2 S protein (GenBank: MN908947), with amino acidexchanges K986P and V987P, was cloned into a starting plasmid vectorwith backbone sequence elements for improved RNA stability andtranslational efficiency (Orlandini von Niessen, A. G. et al., Mol Ther27, 824-836, 2019; Holtkamp, S. et al., Blood 108, 4009-4017, 2006).Non-coding backbone elements included the regions from the T7 promoterto the 5′ and 3′ UTR plus a poly(A) tail (100 nucleotides) interruptedby a linker (A30LA70, 10 nucleotides). The DNA was purified,spectrophotometrically quantified, and in vitro transcribed by T7 RNApolymerase in the presence of a trinucleotide cap1 analogue ((m₂^(7,3′-O))Gppp(m^(2′-O))ApG; TriLink) and ofN1-methylpseudouridine-5′-triphosphate (m1ψTP; Thermo Fisher Scientific)instead of uridine-5′-triphosphate (UTP) (Grudzien-Nogalska, E. et al.,Methods in molecular biology (Clifton, N.J.) 969, 55-72, 2013). RNA waspurified using magnetic particles (Berensmeier, S., Appl. Microbiol.Biotechnol. 73, 495-504, 2006), integrity assessed by microfluidiccapillary electrophoresis (Agilent Fragment Analyser), andconcentration, pH, osmolality, endotoxin level and bioburden determined.

Lipid-Nanoparticle Formulation of the RNA.

Purified RNA was formulated into LNPs using an ethanolic lipid mixtureof ionisable cationic lipid and transferred into an aqueous buffersystem via diafiltration to yield an LNP composition similar to onepreviously described (Maier, M. A. et al., Molecular therapy: thejournal of the American Society of Gene Therapy 21, 1570-1578, 2013).BNT162b2 was stored at −70° C. at a concentration of 0.5 mg/mL.

Immunisation.

Male Rhesus macaques (2-4 years) were randomly assigned to receiveeither BNT162b2 or saline placebo control in 0.5 mL volume administeredby IM injection in the left quadriceps muscle on Days 0 and 21. Bloodfor serum and PBMCs was collected in compliance with animal protocol2017-8725-023 approved by the NIRC Institutional Animal Care and UseCommittee. Animals were anesthetised with ketamine HCl (10 mg/kg; IM)during blood collection and immunisation, and monitored for adequatesedation.

SARS-CoV-2 Challenge of Rhesus macaques.

The SARS-CoV-2 inoculum was obtained from a stock of 2.1×10⁶ PFU/mLpreviously prepared at Texas Biomedical Research Institute (San Antonio,Tex.), aliquoted into single use vials, and stored at −70° C. Theworking virus stock was generated from two passages of the SARS-CoV-2USA-WA1/2020 isolate (a 4^(th) passage seed stock purchased from BEIResources; NR-52281) in Vero E6 cells. The virus was confirmed to beSARS-CoV-2 by deep sequencing and identical to the published sequence(GenBank accession number MN985325.1).

BNT162b2-immunised (n=6) and age-matched saline control-immunised (n=6)male Rhesus macaques (control) were challenged with 1×10⁶ plaque formingunits of SARS-CoV-2 USA-WA1/2020 isolate, split equally between theintranasal (IN; 0.2 mL) and intratracheal (IT; 0.2 mL) routes aspreviously described (Singh, D. K. et al. SARS-CoV-2 infection leads toacute infection with dynamic cellular and inflammatory flux in the lungthat varies across nonhuman primate species (2020)). The challenge wasperformed 41 to 48 days after the second immunisation. A separatesentinel group of non-immunised age- and sex-matched animals (n=3)received only DMEM supplemented with 10% FCS IN (0.2 mL) and IT (0.2mL). Approximately two weeks prior to challenge, animals were moved tothe ABSL-3 facility at Southwest National Primate Research Center(SNPRC; San Antonio, Tex.). Animals were monitored regularly by aboard-certified veterinary clinician for rectal body temperature, weightand physical examination. Specimen collection was performed undertiletamine zolazepam (Telazol) anaesthesia as described (Singh, D. K. etal. SARS-CoV-2 infection leads to acute infection with dynamic cellularand inflammatory flux in the lung that varies across nonhuman primatespecies (2020)). Nasal swabs were collected from macaques at 0, 1, 3,and 6 days after inoculation to assess viral titers. Bronchoalveolarlavage (BAL) was performed the week before challenge and at Days 3 and 6post-inoculation by instilling four times 20 mL of saline. Thesewashings were pooled, aliquoted and stored frozen at −70° C.

Reverse-Transcription Quantitative Polymerase Chain Reaction.

SARS-CoV-2 was detected and quantified in NHP essentially as describedabove in Example 10.

Results

Results showed COVID-19 mRNA Vaccine BNT162b2 was immunogenic elicitingIgG responses after a single dose which were boosted by a second dose.These also showed a dose-response. At 30 μg BNT162, the neutralizinggeometric mean titre was compared to that seen in covalescent plasmafrom human patients with SARS CoV-2 and found to be ^(˜)8-fold higherwith seven days after Dose of the higher dose of 100 μg giving a higherexcess of ^(˜)18-fold and remaining 3.3-times higher than this benchmarkfive weeks after the last immunization. In monkeys the response was alsocharacterised as Th1-dominant with IFN-γ and IL-2, but no IL-4 response.CD4 and CD8 positive cellular responses were also observed in monkeystoo. Such cellular immune response was characterized as a stronglyTh1-biased CD4⁺ T cell response with a concurrent interferon-γ (IFN-γ)+CD8+ T cell response.

The groups of Rhesus macaques (n=6) that had received two immunisationswith 100 μg BNT162b2 or buffer control were challenged 41 to 48 daysafter the second immunisation with 1×10⁶ plaque forming units ofSARS-CoV-2 (strain USA-WA1/2020), split equally between the intranasaland intratracheal routes, as previously described (Singh, D. K. et al.SARS-CoV-2 infection leads to acute infection with dynamic cellular andinflammatory flux in the lung that varies across nonhuman primatespecies, 2020). Three non-immunised, age-matched, male Rhesus macaques(sentinel) were mock-challenged with cell culture medium. At the time ofchallenge, SARS-CoV-2 neutralising titers ranged from 204 to 938 in theBNT162b2-immunised animals and were undetectable in animals from thecontrol-immunised and sentinel groups.

SARS-CoV-2 RNA was measured in bronchoalveolar lavage (BAL) and nasalswab samples by reverse-transcription quantitative polymerase chainreaction (RT-qPCR). All BAL and nasal swab samples obtained before theinfectious challenge and all those obtained from sentinel animals lackeddetectable SARS-CoV-2 RNA (FIG. 68 ). Three days after SARS-CoV-2challenge, viral RNA was detected in BAL fluid from 5/6control-immunised and 2/6 BNT162b2-immunised animals (FIG. 68 ). By 6days after challenge, all six BNT162b2-immunised macaques hadundetectable viral RNA in their lungs; of the control-immunisedmacaques, three had a high level of viral RNA in BAL fluid, two hadcleared, and one had an indeterminate RT-qPCR result. After SARS-CoV-2challenge, viral RNA was detected in nasal swabs of thecontrol-immunised group at each time point: two animals at Day 1, threeanimals at Days 3 and 6 after challenge (FIG. 68 ). InBNT162b2-immunised animals, all nasal swabs were negative at Day 3 andat Day 6.

In lung tissues, control monkeys had evidence of pulmonary diseaseindicated by their increased scores on computed tomography scans with asuggestion of recovery in that scores at day 10 were less than those atday 3; in contrast, the monkeys given COVID-19 mRNA Vaccine BNT162b2 hadlower scores. Microscopic analysis of lung tissues showed that lunginflammation was similar between control and BNT162b2-immunized monkeys,and there was no evidence of enhanced respiratory disease.

Discussion

Results in nonhuman primates confirm the potency and ability ofBNT162b2, an LNP-formulated m1ψ nucleoside-modified mRNA encoding the Santigen captured in a prefusion conformation, to protect againstSARS-CoV-2 challenge in a preclinical model of acute SARS-CoV-2infection.

Example 12: Storage, Shipping and Dose Preparation

This example illustrates storage, shipping and dose preparation of amulti-dose vial of BNT162b2 concentrate for injection.

As shown in FIG. 69 , at the stage of primary packing, 2 ml type 1glass, preservative-free, multi-dose vial (MDV) is used, wherein the MDVhas 0.45 ml frozen liquid drug product and there are 5 doses per vial.At the stage of secondary packing, a single tray holds 195 vials, suchas 975 doses per tray. The tray (white box) dimensions are 229×229×40mm. At the stage of teriary packing, a minimum of 1 tray (975 doses) (orup to 5 trays (max 4875 doses)) is stacked in a payload carton. Thepayload carton is submerged in 23 Kg of dry ice pellets (10 mm-16 mmpellets). The thermal shipper dimensions are as follows: internaldimensions: 245 mm×245 mm×241 mm; external dimensions: 400 mm×400 mm×560mm. The total weight of the thermal shipper is ^(˜)35 Kg.

Different sizes of Ultra-Low Temperature (ULT) freezers are available inthe market. FIG. 70 shows an example for a small volume storage (about90 litres; about 30K doses (left)) and for a large volume storage (about500 litres; about 200K doses (right)). Thermal shipper keeps ULT (e.g.,−90° C. to −60° C.) up to 10 days if stored at 15° C. to 25° C.temperatures without opening and such storage period of time can beextended further by consistently refilling to the top of the containerwith dry ice. Upon receipt and after opening, the box should bereplenished with dry ice within 24 hours (23 Kg of dry ice pellets (10mm-16 mm pellets). Thermal shipper should be re-iced every 5 days. It isrecommended that the thermal shipper is opened not more than twice aday. Thermal shipper should be closed within 1 minute (or less) afteropening. The vaccine can be stored at 2° C. to 8° C. up to 2 days or atroom temperature for no more than 2 hours after thawing. Post-dilutionin use period is 6 hours.

FIG. 71 shows an exemplary dose preparation for a BNT162b2 5-dose vialwhich contains a frozen concentrated solution that is preservative-freeand must be thawed and diluted prior to administration. The preparationsteps are as follows:

Remove a 5-dose vial of BNT162b2 concentrate for injection from itscarton in frozen storage and allow to thaw for approximately 30 minutesat room temperature (e.g., up to 25° C.). In some embodiments, such amulti-dose vial of BNT162b2 may be thawed and stored in a refrigerator(e.g., 2° C.-8° C.), for example, for up to 5 days. Vials thawed at roomtemperature must be diluted within 2 hours or transferred to arefrigerator. Undiluted vials may be stored for up to 48 hours in therefrigerator. Do not refreeze thawed vials. During storage, minimizeexposure to room light, and avoid exposure to direct sunlight andultraviolet light. Thawed vials can be handled in room light conditions.

After thawing and prior to use, ensure the vial is equilibrated to roomtemperature, and invert gently 10 times to mix. Do not shake.

Using aseptic technique, cleanse the vial stopper with a single-useantiseptic swab, then dilute the thawed vial of BNT162b2 by adding 1.8mL of 0.9% Sodium Chloride Injection, USP into the vial. Needles 21gauge or narrower needles are recommended. However, those skill in theart will understand that in some embodiments, wider needles may be used.For example, in some embodiments, needles 20, 19, 18, 17, 16, 15 orwider needles may be used.

You may feel some pressure in the vial as you add the diluent. Ensurevial pressure is equalized by withdrawing 1.8 mL air into the emptydiluent syringe before removing the needle from the vial.

Gently invert the diluted vial 10 times to mix. Do not shake.

Record the date and time of dilution in the appropriate place on theBNT162b2 vial label. Expiry is 6 hours from the time of dilution.Diluted multi-dose vials are stored between 2° C. and 25° C. Do notfreeze. Discard if frozen.

Using aseptic technique, cleanse the vial stopper with a single-useantiseptic swab, and draw up 0.3 mL of the diluted dosing solution intoa new sterile dosing syringe with a needle appropriate for intramuscularinjection. Adjustments to remove air bubbles should be done with theneedle still in the vial to avoid loss of dosing solution. It isrecommended to use the same needle to withdraw and administer the dosewhenever possible. If a second needle is required for administration,pull back on the syringe plunger until a small amount of air enters thesyringe prior to removing the first needle to avoid loss of dosingsolution during the needle change. Take care when priming theadministration needle to prevent any loss of dose.

For each additional dose, use a new sterile syringe and needle andensure the vial stopper is cleansed with antiseptic before eachwithdrawal. Prepared syringes should be administered immediately. Ifthey cannot be administered immediately, they must be administeredwithin 6 hours of the initial vial dilution. Before administration,ensure a final injection volume in the syringe of 0.3 mL.

Example 13: Vaccine Candidate Against COVID-19 Achieved Success in FirstInterim Analysis from Phase 3 Study

The Phase 3 clinical trial of BNT162b2 has enrolled more than 43,000participants to date, almost 39,000 of whom have received a second doseof the vaccine candidate as of Nov. 8, 2020. Approximately 42% of globalparticipants and 30% of U.S. participants have racially (e.g., includingWhite, Black or African American, American Indian or Alaska native,Asian, native Hawaiian or other Pacific Islander, multiracial) andethnically (e.g., including Hispanic/Latino and non-Hispanic/non-Latino)diverse backgrounds. The trial is continuing to enroll and is expectedto continue through the final analysis when a total of 164 confirmedCOVID-19 cases have accrued.

Vaccine candidate BNT162b2 achieved success in First Interim Analysisfrom Phase 3 Study. The vaccine candidate was found to be more than 90%effective in preventing COVID-19 in participants without evidence ofprior SARS-CoV-2 infection in the first interim efficacy analysis.Analysis evaluated 94 confirmed cases of COVID-19 in trial participants.No serious safety concerns have been observed.

A set of results from the Phase 3 COVID-19 vaccine trial, assembled byNov. 4, 2020, provides evidence of the ability of BNT162b2 to preventCOVID-19. The case split between vaccinated individuals and those whoreceived the placebo indicates a vaccine efficacy rate above 90%, at 7days after the second dose. In particular, early analysis of the resultsshowed that individuals who received two injections of the vaccine threeweeks apart experienced more than 90% fewer cases of symptomaticCOVID-19 than those who received a placebo. This confirms thatprotection is achieved 28 days after the initiation of the vaccination,which consists of a 2-dose schedule.

Preliminary such data includes the following tables:

TABLE 5 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days AfterDose 2 - Subjects Without Evidence of Infection Prior to 7 Days AfterDose 2 - Evaluable Efficacy Population (7 Days) - Interim Analysis 1.Vaccine Group (as Randomized) BNT162b2 (30 μg) Placebo (N^(a) = 16061)(N^(a) = 16218) Pr Efficacy Surveillance Surveillance VE (VE >30% |Endpoint n1^(b) Time^(c) (n2^(d)) n1^(b) Time^(c) (n2^(d)) (%) (95%CI^(e)) data)^(f) First COVID-19 4 1.72161 90 1.73212 95.5 (88.8,98.4) >0.9999 occurrence from (15899) (16010) 7 days after Dose 2Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT =nucleic acid amplification test; SARS-CoV-2 = severe acute respiratorysyndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had noserological or virological evidence (prior to 7 days after receipt ofthe last dose) of past SARS-CoV-2 infection (ie, N-binding antibody[serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasalswab] at Visits 1 and 2), and had negative NAAT at any unscheduled visitprior to 7 days after Dose 2 were included in the analysis. Note: Datafrom subjects who are not confirmed 7 days post dose 2 cases areincluded in the analysis to comprehensively show all data reportedand/or contribute to the total surveillance time calculation but may besubject to change with additional follow-up. ^(a)N = number of subjectsin the specified group. ^(b)n1 = Number of subjects meeting the endpointdefinition. ^(c)Total surveillance time in 1000 person-years for thegiven endpoint across all subjects within each group at risk for theendpoint. Time period for COVID-19 case accrual is from 7 days afterDose 2 to the end of the surveillance period. ^(d)n2 = Number ofsubjects at risk for the endpoint. ^(e)Credible interval for VE wascalculated using a beta-binomial model adjusted for surveillance time.^(f)Posterior probability (Pr) was calculated using a beta-binomialmodel adjusted for surveillance time. This probability must be at least99.5% at the interim analysis in order to conclude that the vaccine isefficacious.

TABLE 6 Severe COVID-19 Occurrence After Dose 1 - Dose 1 All- AvailableEfficacy Population - Interim Analysis 1. Vaccine Group (as Randomized)BNT162b2 (30 μg) Placebo (N^(a) = 21617) (N^(a) = 21633) EfficacyEndpoint n^(b) n^(b) Severe COVID-19 occurrence 0 7 after Dose 1 Note:Data from subjects who are not confirmed 7 days post dose 2 cases areincluded in the analysis to comprehensively show all data reportedand/or contribute to the total surveillance time calculation but may besubject to change with additional follow-up. ^(a)N = number of subjectsin the specified group. ^(b)n = Number of subjects meeting the endpointdefinition.

Example 14: Efficacy and Immunogenicity Evaluation Efficacy Results

Vaccine efficacy of BNT162b2 against COVID-19 among participants withoutevidence of past SARS-CoV-2 infection was demonstrated at the firstinterim analysis conducted after accrual of at least 62 cases followingthe protocol and SAP. The primary efficacy results presented in thissection are from that interim analysis.

Only the vaccine efficacy of BNT162b2 for the first primary efficacyendpoint (COVID-19 incidence based on central laboratory or locallyconfirmed NAAT in participants without serological or virologicalevidence of past SARS-CoV-2 infection prior to 7 days after receipt ofthe second dose) is analyzed and presented at this interim analysis.

First Primary Efficacy Endpoint

Among participants included in the evaluable efficacy population, 32,279participants (16,061 in BNT162b2 group and 16,218 in placebo group) didnot have evidence of infection with SARS-CoV-2 through 7 days after thesecond dose. There were 4 COVID-19 cases in the BNT162b2 group comparedto 90 COVID-19 cases reported in the placebo group. These data give anestimated vaccine efficacy of 95.5% for BNT162b2. The posteriorprobability of >99.99% met the prespecified interim analysis successcriterion of >99.5% (Table 7). The 95% credible interval for the vaccineefficacy was 88.8% to 98.4%, indicating that given the current observeddata there is a 95% probability that the true VE lies in this interval.Also, note that the posterior probability that true VE>86.0% is 99.5%and VE>88.8% is 97.5%.

TABLE 7 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days AfterDose 2 - Subjects Without Evidence of Infection Prior to 7 Days AfterDose 2 - Evaluable Efficacy Population (7 Days) - Interim Analysis 1Vaccine Group (as Randomized) BNT162b2 (30 μg) Placebo (N^(a) = 16061)(N^(a) = 16218) Pr Efficacy Surveillance Surveillance VE (VE >30% |Endpoint n1^(b) Time^(c) (n2^(d)) n1^(b) Time^(c) (n2^(d)) (%) (95%CI^(e)) data)^(f) First COVID-19 4 1.722 90 1.732 95.5 (88.8,98.4) >0.9999 occurrence (15899) (16010) from 7 days after Dose 2Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT =nucleic acid amplification test; SARS-CoV-2 = severe acute respiratorysyndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had noserological or virological evidence (prior to 7 days after receipt ofthe last dose) of past SARS-CoV-2 infection (ie, N-binding antibody[serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasalswab] at Visits 1 and 2), and had negative NAAT at any unscheduled visitprior to 7 days after Dose 2 were included in the analysis. Note: Datafrom subjects who are not confirmed 7 days post dose 2 cases areincluded in the analysis to comprehensively show all data reportedand/or contribute to the total surveillance time calculation but may besubject to change with additional follow-up. ^(a)N = number of subjectsin the specified group. ^(b)n1 = Number of subjects meeting the endpointdefinition. ^(c)Total surveillance time in 1000 person-years for thegiven endpoint across all subjects within each group at risk for theendpoint. Time period for COVID-19 case accrual is from 7 days afterDose 2 to the end of the surveillance period. ^(d)n2 = Number ofsubjects at risk for the endpoint. ^(e)Credible interval for VE wascalculated using a beta-binomial model adjusted for surveillance time.^(f)Posterior probability (Pr) was calculated using a beta-binomialmodel adjusted for surveillance time. This probability must be at least99.5% at the interim analysis in order to conclude that the vaccine isefficacious.

The vaccine efficacy of BNT162b2 for the same primary efficacy endpointbased on the all-available efficacy population was 95.7%, with 4 and 93cases in the BNT162b2 and placebo groups, respectively.

No clinically meaningful differences in VE by subgroup were observed byage group, country, ethnicity, sex, or race in the in the Dose 2evaluable efficacy population, with VE estimates that ranged from 91.2%to 100.0% (Table 8).

TABLE 8 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days AfterDose 2, by Subgroup - Subjects Without Evidence of Infection Prior to 7Days After Dose 2 - Evaluable Efficacy Population (7 Days) - InterimAnalysis 1 Vaccine Group (as Randomized) BNT162b2 (30 μg) PlaceboEfficacy (N^(a) = 16061) (N^(a) = 16218) Endpoint SurveillanceSurveillance Subgroup n1^(b) Time^(c) (n2^(d)) n1^(b) Time^(c) (n2^(d))VE (%) (95% CI^(e)) First COVID-19 occurrence from 7 days after Dose 2Overall 4 1.722 (15899) 90 1.732 (16010) 95.5 (88.1, 98.8) Age group(years) 16 to 55 2 0.954 (8994) 67 0.959 (9040) 97.0 (88.7, 99.6) >55 20.767 (6905) 23 0.773 (6970) 91.2 (64.6, 99.0) Sex Male 2 0.874 (8115)38 0.865 (8029) 94.8 (79.8, 99.4) Female 2 0.848 (7784) 52 0.867 (7981)96.1 (85.1, 99.5) Race White 4 1.477 (13399) 85 1.491 (13530) 95.3(87.4, 98.7) Black or African 0 0.124 (1263) 4 0.124 (1277) 100.0(−51.8, 100.0) American All others^(f) 0 0.121 (1237) 1 0.118 (1203)100.0 (−3690.1, 100.0)  Ethnicity Hispanic/Latino 1 0.464 (4389) 340.459 (4342) 97.1 (82.7, 99.9) Non-Hispanic/ 3 1.247 (11418) 56 1.262(11570) 94.6 (83.3, 98.9) non-Latino Country Argentina 0 0.271 (2436) 280.266 (2402) 100.0  (86.2, 100.0) Brazil 0 0.087 (878) 2 0.087 (879)100.0 (−432.5, 100.0)  USA 4 1.360 (12384) 60 1.376 (12530) 93.3 (81.8,98.2) Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT= nucleic acid amplification test; SARS-CoV-2 = severe acute respiratorysyndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had noserological or virological evidence (prior to 7 days after receipt ofthe last dose) of past SARS-CoV-2 infection (ie, N-binding antibody[serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasalswab] at Visits 1 and 2), and had negative NAAT at any unscheduled visitprior to 7 days after Dose 2 were included in the analysis. Note: Datafrom subjects who are not confirmed 7 days post dose 2 cases areincluded in the analysis to comprehensively show all data reportedand/or contribute to the total surveillance time calculation but may besubject to change with additional follow-up. ^(a)N = number of subjectsin the specified group. ^(b)n1 = Number of subjects meeting the endpointdefinition. ^(c)Total surveillance time in 1000 person-years for thegiven endpoint across all subjects within each group at risk for theendpoint. Time period for COVID-19 case accrual is from 7 days afterDose 2 to the end of the surveillance period. ^(d)n2 = Number ofsubjects at risk for the endpoint. ^(e)Confidence interval (CI) for VEis derived based on the Clopper and Pearson method adjusted to thesurveillance time. ^(f)American Indian or Alaska native, Asian, NativeHawaiian or other Pacific Islander, multiracial, not reported racecategories are presented as “All others”.

Severe COVID-19 Cases

Severe COVID-19 cases were reported in a total of 7 participants inPhase 3, all in the placebo group, as of the data cutoff date of 4 Nov.2020 for the first interim analysis (Table 9). Five of these cases werereported between Dose 1 and Dose 2, none were reported less than 7 daysafter Dose 2, and 2 cases were reported at least 7 days after Dose 2.

TABLE 9 Severe COVID-19 Occurrence After Dose 1 - Dose 1 All- AvailableEfficacy Population - Interim Analysis 1 Vaccine Group (as Randomized)BNT162b2 (30 μg) Placebo (N^(a) = 21617) (N^(a) = 21633) EfficacyEndpoint n^(b) n^(b) Severe COVID-19 occurrence 0 7 after Dose 1 Note:Data from subjects who are not confirmed 7 days post dose 2 cases areincluded in the analysis to comprehensively show all data reportedand/or contribute to the total surveillance time calculation but may besubject to change with additional follow-up. ^(a)N = number of subjectsin the specified group. ^(b)n = Number of subjects meeting the endpointdefinition.

Efficacy Conclusions

The first primary efficacy objective met success criteria. BNT162b2achieved vaccine efficacy of 95.5% with a 2-sided 95% credible intervalof 88.8% to 98.4% among participants without evidence of infection up to7 days after Dose 2, and a >99.99% posterior probability for the truevaccine efficacy greater than 30% conditioning on available data.

All 7 severe COVID-19 cases (after Dose 1) were observed in the placebogroup, as of the interim analysis cutoff date.

Immunogenicity Results Phase 1

This Phase 1 interim Clinical Study Report (CSR) presents immunogenicityresults for both adult age groups up to 1 month after Dose 2 for theBNT162b1 and BNT162b2 vaccine candidates at the 10-μg, 20-μg, and 30-μgdose levels, and up to 7 weeks after Dose 1 of BNT162b1 at the 100-μgdose level (younger age group only).

Results for the 7 days after Dose 1 time point are only analyzed andpresented in the younger age group (18 to 55 years of age) for 10 μg and30 μg BNT162b1.

SARS-CoV-2 Neutralizing Titers—Phase 1 GMTs

Overall, for both the BNT162b1 and the BNT162b2 recipients in both agegroups, SARS-CoV-2 50% neutralizing GMTs modestly increased by Day 21after Dose 1 and were substantially increased 7 days after Dose 2.Generally, GMTs in the older age group tended to be somewhat lower thanthe GMTs in the younger age group at most time points for both BNT162b1and BNT162b2 recipients.

BNT162b1

In the younger age group, SARS-CoV-2 50% neutralizing GMTs modestlyincreased by Day 21 after Dose 1 and were substantially increased 7 daysafter Dose 2 (Day 28) of BNT162b1, with higher GMTs observed in the30-μg dose group compared to the 10-μg and 20-μg dose groups (FIG. 72 ).GMTs increased at 14 days after Dose 2 (Day 35) for all dose groups, andalthough GMTs decreased at 1 month after Dose 2 (Day 52), the Day 52GMTs remained substantially higher than those at the earlier time pointsafter Dose 1.

In the 100-μg dose group, SARS-CoV-2 50% neutralizing GMTs modestlyincreased by Day 21 after Dose 1 of BNT162b1 and decreased to a nearbaseline value by Day 52. Generally similar trends were observed in theolder age group, with higher GMTs observed in the 20-μg and 30-μg dosegroups of BNT162b1 compared to the 10-μg dose group (FIG. 73 ).

Similar trends were observed for the SARS-CoV-2 90% neutralizing GMTs.

Results for the all-available immunogenicity population in the youngerage and older age groups were similar to those observed for theevaluable immunogenicity population.

RCDCs of SARS-CoV-2 50% and 90% neutralizing titers after BNT162b1 forthe younger and older age groups show that the majority of participantsresponded by 7 days after Dose 2 of BNT162b1.

BNT162b2

In the younger age group, SARS-CoV-2 50% neutralizing GMTs increased byDay 21 after Dose 1 and were substantially increased 7 days after Dose 2(Day 28) of BNT162b2, with higher GMTs observed in the 20-μg and 30-μgdose groups compared to the 10-μg dose group (FIG. 74 ). The GMTsdecreased at 14 days after Dose 2 (Day 35) and 1 month after Dose 2 (Day52) of BNT162b2; however, the GMTs remained substantially higher thanthose at the earlier time points after Dose 1. Similar trends weregenerally observed in the older age group, with higher GMTs observed inthe 30-μg dose groups compared to the 20-μg and 10-μg dose groups (FIG.75 ). SARS-CoV-2 50% neutralizing GMTs were increased 7 days after Dose2 and were similar in the 10-μg and 20-μg dose groups and higher in the30-μg dose group. At 1 month after Dose 2, GMTs remained substantiallyhigher than those at the earlier time points after Dose 1. In the olderage group, SARS-CoV-2 50% neutralizing GMTs were generally lower thanthe GMTs in the younger age group. Similar trends were observed for theSARS-CoV-2 90% neutralizing GMTs.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population. RCDCs of SARS-CoV-2 50% and 90% neutralizingtiters for the younger and older age groups show that the majority ofparticipants responded by 7 days after Dose 2 of BNT162b2.

GMFRs

Overall, for both the BNT162b1 and the BNT162b2 recipients, and in bothage groups, GMFRs of SARS-CoV-2 50% neutralizing titers from beforevaccination to 7 days after Dose 2 (Day 28) were substantially highercompared to the respective GMFRs after Dose 1. GMFRs in the older agegroup were generally lower than the those in the younger age group forboth BNT162b1 and BNT162b2 recipients.

BNT162b1

In the younger age group, GMFRs of SARS-CoV-2 50% neutralizing titersfrom before vaccination to 7 days after Dose 2 (Day 28) of BNT162b1 weresubstantially high compared to GMFRs at earlier time points after Dose 1of BNT162b1 in all dose groups, with GMFRs being highest in the 30-μgdose group. At 1 month after Dose 2, the GMFRs remained higher thanthose at the earlier time points after Dose 1.

In the 100-μg dose group, the GMFRs of SARS-CoV-2 50% neutralizingtiters were not substantially increased through Day 52 after Dose 1 ofBNT162b1.

In the older age group, GMFRs of SARS-CoV-2 50% neutralizing titers frombefore vaccination to 7 days after Dose 2 (Day 28) of BNT162b1 weresubstantially high compared to GMFRs at the earlier time point afterDose 1 of BNT162b1 in the 20-μg and 30-μg dose groups with GMFRs beinghighest in the 20-μg dose group. The GMFRs remained high in the 20-μgand 30-μg dose groups at 1 month after Dose 2 (Day 52) of BNT162b1compared to GMFRs at the earlier time point after Dose 1.

Similar trends were observed for GMFRs of SARS-CoV-2 90% neutralizingtiters in the younger age group and older age group.

Results for the all-available immunogenicity population in the youngerage and older age groups were similar to those observed for theevaluable immunogenicity population.

BNT162b2

In the younger age group, GMFRs of SARS-CoV-2 50% neutralizing titersfrom before vaccination to 7 days after Dose 2 (Day 28) of BNT162b2 weresubstantially high compared to GMFRs at the earlier time point afterDose 1 of BNT162b2 for all dose groups, with GMFRs being similar andhighest in the 20-μg and 30-μg dose groups. GMFRs remained high through1 month after Dose 2 of BNT162b2 compared to GMFRs 21 days after Dose 1of BNT162b2.

In the older age group, GMFRs of SARS-CoV-2 50% neutralizing titers frombefore vaccination to 7 days after Dose 2 (Day 28) of BNT162b2 weresubstantially high compared to GMFRs at the earlier time point afterDose 1 of BNT162b2 in all dose groups, with GMFRs being highest in the30-μg dose group. GMFRs remained high through 1 month after Dose 2 ofBNT162b2 compared to GMFRs at 21 days after Dose 1 of BNT162b2.

Similar trends were observed for GMFRs of SARS-CoV-2 90% neutralizingtiters in the younger and older age groups.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

Number (%) of Participants Achieving a ≥4-Fold Rise

Overall, for both the BNT162b1 and the BNT162b2 recipients, and in bothage groups, most participants achieved a ≥4-fold rise in SARS-CoV-2 50%neutralizing titers from before vaccination to 7 days after Dose 2,except in the older participants in the 10-μg BNT162b1 dose group.

BNT162b1

In the younger age group, from before vaccination to 21 days after Dose1 of BNT162b1, no participants in the 10-μg dose group and 53participants in the 20-μg and the 30-μg dose groups achieved a ≥4-foldrise in SARS-CoV-2 50% neutralizing titers. From before vaccination toboth 7 days and 1 month after Dose 2 of BNT162b1 most or allparticipants in the 10-μg, 20-μg, and 30-μg dose groups achieved a≥4-fold rise in SARS-CoV-2 50% neutralizing titers.

In the older age group, from before vaccination to 21 days after Dose 1of BNT162b1, only 1 participant in the 30-μg dose group achieved a≥4-fold rise in SARS-CoV-2 50% neutralizing titers. From beforevaccination to both 7 days and 1 month after Dose 2 of BNT162b1, 52participants in the 10-μg group and 9 to 11 participants in the 20-μgand 30-μg dose groups achieved a ≥4-fold rise in SARS-CoV-2 50%neutralizing titers.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

BNT162b2

In the younger age group, from before vaccination to 21 days after Dose1 of BNT162b2, 2 (18.2%) participants in the 10-μg dose group, 3 (25.0%)participants in the 20-μg dose group, and none in the 30-μg groupachieved a ≥4-fold rise in SARS-CoV-2 50% neutralizing titers. Frombefore vaccination to 7 days after Dose 2 of BNT162b2, all participantsachieved a ≥4-fold rise in SARS-CoV-2 50% neutralizing titers, which wasmaintained through 1 month after Dose 2 of BNT162b2.

In the older age group, from before vaccination to 21 days after Dose 1of BNT162b2, no participants achieved a 4-fold rise in SARS-CoV-2 50%neutralizing titers in any dose group. From before vaccination to 7 daysafter Dose 2 of BNT162b2, 10 (83.3%), 9 (81.8%), and 10 (90.9%)participants achieved a 4-fold rise in SARS-CoV-2 50% neutralizingtiters in the 10-μg, 20-μg, and 30-μg dose groups, respectively. Frombefore vaccination to 1 month after Dose 2 of BNT162b2, 9 (75.0%), 6(54.5%), and 11 (100.0%) participants achieved a ≥4-fold rise inSARS-CoV-2 50% neutralizing titers in the 10-μg, 20-μg, and 30-μg dosegroups, respectively.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

SARS-CoV-2 Antigen-Specific Binding Antibody Levels—Phase 1

Vaccine candidate BNT162b1 encodes for the RBD of SARS-CoV-2.RBD-binding IgG responses for each dose level and age group for thiscandidate are described in this section. RBD-binding IgG levels werealso assessed for candidate BNT62b2 which encodes the P2 S ofSARS-CoV-2.

Vaccine candidate BNT162b2 encodes for the P2 S of SARS-CoV-2.S1-binding IgG responses for each dose level and age group for thiscandidate are described in this section. S1-binding IgG levels were alsoassessed for candidate BNT62b1 which encodes the RBD of SARS-CoV-2.

GMCs

Overall, for both the BNT162b1 and the BNT162b2 recipients, and in bothage groups, RBD- and S1-binding GMCs increased substantially by Day 21after Dose 1 and were further increased 7 days after Dose 2. Responseswere maintained through Day 52. GMCs in the older age group weregenerally lower than the GMCs in the younger age group, with theexception of Day 28 in the 20-μg BNT162b1 dose group for both RBD- andS1-binding IgG levels.

BNT162b1

In the younger age group, RBD-binding GMCs increased substantially byDay 21 after Dose 1 of BNT162b1 and further increased 7 days after Dose2 (Day 28) of BNT162b1, with higher GMCs observed in the 30-μg dosegroup compared to the 10-μg and 20-μg dose groups (FIG. 76 ). At 1 monthafter Dose 2 (Day 52), the GMCs remained substantially higher than atthe earlier time points after Dose 1.

In the 100-μg BNT162b1 group, the RBD-binding GMC increasedsubstantially by 21 days after BNT162b1 and remained higher through Day52 compared to the Day 7 GMC.

In the older age group, RBD-binding GMCs increased substantially by Day21 after Dose 1 of BNT162b1 and further increased 7 days after Dose 2(Day 28) of BNT162b1, with higher GMCs observed in the 20-μg and 30-μgdose groups compared to the 10-μg group (FIG. 77 ). At 1 month afterDose 2 (Day 52), the GMCs remained substantially higher than at theearlier time point after Dose 1.

S1-binding IgG GMC results for BNT162b1 were similar to those observedfor RBD-binding IgG GMCs in the younger (FIG. 78 ) and older age groups(FIG. 79 ), and in the 100-μg BNT162b1 group.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population. RCDCs of RBD- and S1-binding IgG levels showthat the majority of participants responded by 21 days after Dose 1 ofBNT162b1.

BNT162b2

In the younger age group, S1-binding GMCs increased substantially by Day21 after Dose 1 of BNT162b2 and were substantially increased by 7 daysafter Dose 2 (Day 28) of BNT162b2, with higher GMCs observed in the20-μg and 30-μg dose groups compared to the 10-μg dose group (FIG. 80 ).At 1 month after Dose 2 (Day 52), the GMCs remained substantially higherthan at the earlier time point after Dose 1.

Similar trends were observed in the older age group, with higherS1-binding GMCs observed in the 30-μg dose group compared to the 10-μgand 20-μg dose groups (FIG. 81 ).

RBD-binding IgG GMC results for BNT162b2 were similar to those observedfor S1-binding IgG GMCs in the younger (FIG. 82 ) and older age groups(FIG. 83 ). Results for the all-available immunogenicity population inthe younger and older age groups were similar to those observed for theevaluable immunogenicity population. RCDCs of RBD- and S1-binding IgGlevels after BNT162b2 show that the majority of participants respondedby 21 days after Dose 1 of BNT162b2.

GMFRs

Overall, for the BNT162b1 and the BNT162b2 recipients, and in both agegroups, GMFRs of RBD-binding IgG levels and GMFRs of S1-binding IgGlevels were substantially high from before vaccination to 21 days afterDose 1, with greater GMFRs observed from before vaccination to 7 daysafter Dose 2.

BNT162b1

GMFRs of RBD-binding IgG levels were substantially high from beforevaccination to Day 21 (before Dose 2) after Dose 1 of BNT162b1, withgreater GMFRs observed from before vaccination to 7 days after Dose 2(Day 28) of BNT162b1 in both the younger and older age groups, for the10-μg, 20-μg, and 30-μg dose groups. GMFRs remained substantially highin the 10-μg, 20-μg, and 30-μg BNT162b1 groups from before vaccinationto 1 month after Dose 2 compared to the earlier time points after Dose 1for both age groups.

In the 100-μg BNT162b1 group, GMFR of RBD-binding IgG levels wassubstantially high from before vaccination by 21 days after BNT162b1 andremained higher through Day 52 compared to the Day 7 GMFR.

Similar trends were observed for GMFRs of S1-binding IgG levels forBNT162b1. Results for the all-available immunogenicity population in theyounger age and older age groups were similar to those observed for theevaluable immunogenicity population.

BNT162b2

GMFRs of S1-binding IgG levels were substantially high from beforevaccination to Day 21 (before Dose 2) after Dose 1 of BNT162b2, withgreater GMFRs observed from before vaccination to 7 days after Dose 2(Day 28) of BNT162b2 in both the younger and older age groups, for the10-μg, 20-μg, and 30-μg dose groups. GMFRs remained substantially highin all BNT162b2 groups from before vaccination to 1 month after Dose 2compared to the earlier time point after Dose 1 for both age groups.

Similar trends were observed for GMFRs of RBD-binding IgG levels forBNT162b2. Results for the all-available immunogenicity population in theyounger and older age groups were similar to those observed for theevaluable immunogenicity population.

Number (%) of Participants Achieving a ≥4-Fold Rise

Overall, for the BNT162b1 and the BNT162b2 recipients, and in both agegroups, all participants achieved a ≥4-fold rise in S1- and RBD-bindingIgG levels from before vaccination to 7 days after Dose 2, with theexception of 1 participant in the younger 20-μg BNT162b1 group.

BNT162b1

In the younger age group, from before vaccination to 21 days followingDose 1 of BNT162b1, all participants (except 1 in the 20-μg dose group)across all dose groups achieved a ≥4-fold rise in RBD-binding IgGlevels. All participants in the 20-μg dose group achieved a ≥4-fold risein RBD-binding IgG levels from before vaccination to 14 days after Dose2 (Day 35).

In the older age group, from before vaccination to 21 days followingDose 1 of BNT162b1, all participants in the 20-μg and 30-μg dose groupsand 8 (72.7%) participants in the 10-μg dose group achieved a ≥4-foldrise in RBD-binding IgG levels. All participants in the 10-μg dose groupachieved a ≥4-fold rise in RBD-binding IgG levels from beforevaccination to 7 days after Dose 2 (Day 28).

Similar trends were generally observed for participants achieving a≥4-fold rise in S1-binding IgG levels for BNT162b1.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

BNT162b2

In the younger age group, from before vaccination to 21 days followingDose 1 of BNT162b2, all participants in each dose group achieved a≥4-fold rise in S1-binding IgG levels.

In the older age group, from before vaccination to 21 days followingDose 1 of BNT162b2, all participants in the 10-μg, and 30-μg dose groupsand 11 (91.7%) participants in the 20-μg dose group achieved a ≥4-foldrise in S1-binding IgG levels. All participants in the 20-μg dose groupachieved a ≥4-fold rise in S1-binding IgG levels from before vaccinationto 7 days after Dose 2 (Day 28).

Similar trends were generally observed for participants achieving a≥4-fold rise in RBD-binding IgG levels for BNT162b2.

Results for the all-available immunogenicity population were similar tothose observed for the evaluable immunogenicity population in theyounger and older age groups.

GMRs of SARS-CoV-2-Neutralizing Titers to SARS-CoV-2 Antigen-SpecificBinding Antibody Levels

Overall, for BNT162b1 and BNT162b2 recipients, GMRs of SARS-CoV-2 50%neutralizing titers to RBD- or S1-binding IgG levels show a more robustRBD- or S1-binding levels relative to neutralizing titers, which weresimilar within each age group.

BNT162b1

At 21 days after Dose 1 at 10 μg, 20 μg, or 30 μg, GMRs of SARS-CoV-250% neutralizing titers to RBD-binding IgG levels were ≤0.035 in theyounger age group and ≤0.183 in the older age group. At 14 days afterDose 2, the GMRs were ≤0.032 in the younger age group and ≤0.018 in theolder age group.

For the 100-μg dose group, the GMR was 0.018 at 21 days after Dose 1 and0.014 at 35 days after Dose 1.

GMRs of SARS-CoV-2 50% neutralizing titers to S1-binding IgG levels weresimilar to GMRs of SARS-CoV-2 50% neutralizing titers to RBD-binding IgGlevels in the younger and older age groups after BNT162b1.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

BNT162b2

At 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers toS1-binding IgG levels were 0.035 in the younger age group and ≤0.124 inthe older age group. At 14 days after Dose 2, the GMRs were ≤50.040 inthe younger age group and ≤0.037 in the older age group.

Results for the all-available immunogenicity population in the youngerand older age groups were similar to those observed for the evaluableimmunogenicity population.

Evaluating BNT162b1 and BNT162b2 GMRs

In the younger age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50%neutralizing titers to RBD-binding IgG levels were 50.035 after BNT162b1and ≤0.054 after BNT162b2. At 14 days after Dose 2, the GMRs were ≤0.032after BNT162b1 and ≤0.046 after BNT162b2.

In the older age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50%neutralizing titers to RBD-binding IgG levels were ≤0.183 after BNT162b1and ≤0.196 after BNT162b2. At 14 days after Dose 2, the GMRs were ≤0.018after BNT162b1 and ≤0.043 after BNT162b2.

In the younger age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50%neutralizing titers to S1-binding IgG levels were ≤0.061 after BNT162b1and ≤0.035 after BNT162b2. At 14 days after Dose 2, the GMRs were ≤0.035after BNT162b1 and ≤0.040 after BNT162b2.

In the older age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50%neutralizing titers to S1-binding IgG levels were ≤0.328 after BNT162b1and ≤0.124 after BNT162b2. At 14 days after Dose 2, the GMRs were ≤0.022after BNT162b1 and ≤0.037 after BNT162b2.

Phase 1 Summary of Immunogenicity Results Evaluating BNT162b1 andBNT162b2

In general, a modest neutralizing immune response was observed in boththe younger and older age groups after the first dose. A much morerobust immune response was observed 7 days after the second dose ofeither BNT162b1 or BNT162b2 at all dose levels in both the younger andolder age groups. Antibody levels at the last time point tested werestill substantially above those at baseline.

In the Younger Age Group:

At 7 days after Dose 2, SARS-CoV-2 50% neutralizing GMTs in the 20-μgand 30-μg dose groups were higher for BNT162b2 recipients than forBNT162b1 recipients. The GMTs were similar in the 10-μg dose group forboth recipients. At 1 month after Dose 2 (Day 52), GMTs remainedsubstantially higher than those at the earlier time points after Dose 1for both BNT162b1 and BNT162b2 recipients.

From before vaccination to 7 days after Dose 2, GMFRs of SARS-CoV-2 50%neutralizing titers were substantially high for BNT162b1 and BNT162b2recipients at the 30 μg dose level.

From before vaccination to 7 days after Dose 2, all participants at the30-μg dose level who received BNT162b1 or BNT162b2 achieved a ≥4-foldrise in SARS CoV-2 50% neutralizing titers.

In the Older Age Group:

At 7 days after Dose 2, SARS-CoV-2 50% neutralizing GMT in the 30-μgdose group was higher for BNT162b2 recipients than for BNT162b1recipients. At 1 month after Dose 2 (Day 52), the SARS-CoV-2 50%neutralizing GMTs in the 30-μg dose group were similar for both BNT162b1and BNT162b2 recipients.

From before vaccination to 7 days after Dose 2, the GMFR of SARS-CoV-250% neutralizing titers were substantially high for BNT162b1 andBNT162b2 recipients at the 30-μg dose level.

From before vaccination to 7 days after Dose 2, most participants whoreceived BNT162b1 or BNT162b2 at the 30-μg dose level achieved a ≥4-foldrise in SARS-CoV-2 50% neutralizing titers.

Phase 1 Immunogenicity Conclusions

Both BNT162b1 and BNT162b2 elicited robust SARS-CoV-2 neutralizingantibody response 7 days after Dose 2 in younger and older adults, basedon GMTs, GMFRs, proportions of participants achieving a ≥4-fold rise inneutralizing titers, and RCDCs. Neutralizing antibody response wasmaintained through Day 52 and was similar for the candidates within thecorresponding age and dose groups.

Both BNT162b1 and BNT162b2 elicited substantial rises in antigen bindingIgG levels 7 days after Dose 2, based on GMCs, GMFRs, and proportions ofparticipants achieving a ≥4-fold rise in IgG-antigen specific binding.Responses were maintained through Day 52.

In the 100-μg dose group, SARS-CoV-2 neutralizing antibody responsemodestly increased by 3 weeks after Dose 1 of BNT162b1, but neutralizingantibody response returned to levels similar to baseline by 7 weeksafter Dose 1.

These data support the need for a 2-dose vaccination series.

Phase 2

Immunogenicity is an exploratory endpoint for the Phase 2 part of thestudy.

Phase 3

Immunogenicity is a secondary (12 to 15 year olds compared with 16 to 25year olds) and an exploratory endpoint for the Phase 3 part of thestudy.

Example 15: Safety Evaluation

In this interim CSR, all participants in Phase 1 and the first 6610participants in Phase 2/3 (360 participants from Phase 2 included) usedan e-diary for reporting local reactions and systemic events. A total of1125 participants in Phase 2/3 were identified as baseline SARS-CoV-2positive, defined as having a positive N-binding antibody test result orpositive nucleic acid amplification test (NAAT) result on the day ofDose 1; of these, 545 received BNT162b2 and 580 received placebo.

Phase 1

Safety data are available up through the date cutoff date (24 Aug. 2020)and are summarized at various time points relative to Dose 1 or Dose 2.Safety results for Phase 1 vaccine candidates BNT162b1 and BNT162b2 forboth adult age groups are presented up to 1 month after Dose 2 (or datacutoff date) at the 10-μg, 20-μg, and 30-μg dose levels. Safety resultsfor BNT162b1 at the 100-μg dose level in the younger age group arepresented up to 3 weeks after Dose 1 or to before Dose 2 based on thedata cutoff date. Note that the group of participants 18 to 55 years ofage who received 100 μg BNT162b1 did not receive a second dose of 100 μgBNT162b2 per IRC decision.

Local Reactions—Phase 1

Overall, for both the BNT162b1 and the BNT162b2 recipients, and in bothage groups, pain at the injection site was the most frequent localreaction. Redness and swelling occurred less frequently in the BNT162b2group and in the BNT162b1 group. In both the BNT162b1 and BNT162b2groups, the frequency of local reactions was lower in the older agegroup compared to the younger age group, and there was a trend of ahigher frequency of local reactions with increased dose.

BNT162b1

In the younger age group, pain at the injection site was the mostfrequently reported local reaction within 7 days after Dose 1 ofBNT162b1. As dose level increased from 10 μg to 30 μg, increasingfrequencies of pain at the injection site (58.3% to 100.0%, 7 and 12participants, respectively) were observed compared to none in theplacebo group (FIG. 84 ). Redness was reported in 2 (16.7%) participantsin the 30-μg dose group, and swelling was reported in 3 (25.0%)participants in the 20-μg dose group and 2 (16.7%) participants in the30-μg dose group. In the 100-μg dose group, pain at the injection site(12 [100.0%] participants), swelling (5 [41.7%] participants), andredness (4 [33.3%] participants) were reported, and 1 [8.3%] participanthad severe injection site pain (note: per IRC decision, Dose 2 was lateradministered to participants at the 10-μg dose level).

Within 7 days after Dose 2 of BNT162b1 in the younger age group, pain atthe injection site remained the most frequently reported local reactionreaching 12 (100.0%) participants with the 30-μg dose group compared tothe placebo group (2 [22.2%] participants), while the proportions ofparticipants with redness (2 [16.7%]participants) and swelling (3[25.0%] participants) were highest in the 30-μg dose group (FIG. 84 ).No redness or swelling was reported in the placebo group.

In the older age group, pain at the injection site was the mostfrequently reported local reaction within 7 days after Dose 1 ofBNT162b1 in both the 20-μg and 30-μg dose groups (11 [91.7%]participants each) compared to the placebo group (1 [11.1%] participant)(FIG. 85 ). No redness was reported, and the maximal frequency ofswelling (2 [16.7%] participants) was in the 30-μg group. No redness orswelling was reported in the placebo group.

Within 7 days after Dose 2 of BNT162b1 in the older age group, pain atthe injection site was the most frequently reported local reaction inboth the 20-μg and 30-μg dose groups (9 [75.0%] participants each). Thefrequency of swelling (3 [25.0%] participants) was maximal at 30 μg,while redness (1 [8.3%] participant each) was reported in the 20-μg and30-μg dose groups. No redness or swelling was reported in the placebogroup.

After the first and second dose and in both age groups, the majority oflocal reactions were mild or moderate in severity, and no Grade 4 localreactions were reported. Overall, for BNT162b1 recipients and in bothage groups, pain at the injection site was the most frequent localreaction (58.3% to 100.0%), and redness (0% to 16.7%) and swelling (0%to 25.0%) occurred at a lower frequency. Notably, the frequency of localreactions was lower in the older age group compared to the younger agegroup, and there was a trend of a higher frequency of local reactionswith increased dose.

In the younger age group, pain at the injection site had median onsetday of Day 1.0 (day of vaccination) after either dose of BNT162b1 acrossdoses 10 μg to 30 μg and after Dose 1 of BNT162b1 100 μg. Median onsetday for redness and swelling was between Day 1.0 and Day 3.0 in all dosegroups.

In the older age group, pain at the injection site had median onset dayof Day 1.0 (day of vaccination) after Dose 1 of BNT162b1 across all dosegroups and after Dose 2 for the 20-μg and 30-μg dose groups (medianonset day was on Day 1.5 in the 10-μg dose group after Dose 2). With theexception of redness on Day 4 (20-μg dose group) and Day 5 (30-μg dosegroup) in 1 participant each after Dose 2, all other local reactions ofredness or swelling reported had median onset day between Day 1.0 andDay 3.0 for all dose groups.

Local reactions resolved with median durations between 1.0 and 4.0 daysin the younger age group and older age group across dose levels.

BNT162b2

In the younger age group, pain at the injection site was the mostfrequently reported local reaction within 7 days after Dose 1, which wasmaximal in the 30-μg dose group (11 [91.7%] participants) (FIG. 86 ).One [8.3%] participant had severe injection site pain after Dose 1 of 30μg. Most participants did not report swelling and redness. After Dose 2,pain at the injection site remained the most frequently reported localreaction (83.3%, 10 participants in each) in the 20-μg and 30-μg dosegroups compared to the placebo group (2 [22.2%] participants). Noparticipants reported redness and swelling for any dose group includingplacebo.

In the older age group, pain at the injection site was reported within 7days after Dose 1 of BNT162b2 in all dose groups and was maximal in the30-μg dose group (75.0%, 9 participants), while no redness and swellingwas reported in any group (FIG. 87 ). Local reactions were not reportedin the placebo group. After Dose 2, pain at the injection site (8[66.7%] participants) was reported in the 30-μg group compared to theplacebo group (9 [11.1%] participants); no participants who receivedBNT162b2 or placebo reported redness and swelling.

After the first and second dose and in both age groups, the majority oflocal reactions were mild or moderate in severity, and no Grade 4 localreactions were reported. Overall, for BNT162b2 recipients and in bothage groups, pain at the injection site was the most frequent localreaction (33.3% to 91.7%), and redness (0% to 8.3%) and swelling (0% to16.7%) were infrequent. The frequency of local reactions was lower inthe older age group compared to the younger age group, and there was atrend of a higher frequency of local reactions with increased dose.

In the younger age group, median onset day for local reactions occurredbetween Day 1.0 (day of vaccination) to Day 2.0 after any dose ofBNT162b2 across any dose level. In the older age group, median onset dayfor local reactions occurred between Day 1.0 (day of vaccination) to Day2.0 after any dose of BNT162b2 across any dose level. Local reactionsgenerally resolved with median durations between 1.0 to 2.0 days in theyounger and older age groups across dose levels.

Systemic Events—Phase 1

Overall, within 7 days after Dose 1, fatigue was generally the mostfrequently reported systemic event in the both the younger and olderBNT162b1 groups and in the older BNT162b2 group; while headache andfatigue were most frequently reported in the younger BNT162b2 dosegroup. Overall, within 7 days after Dose 2, headache was the mostfrequently reported systemic event in the both the younger and olderBNT162b1 groups and fatigue was the most frequently reported systemicevent in the both the younger and older BNT162b2 groups. Chills wasgenerally reported at a higher frequency after Dose 2 and at a higherfrequency in the BNT162b1 group than in the BNT162b2 group. Fever wasreported more frequently in the younger BNT162b1 group after Dose 2 thanin the older BNT162b2 group. For both the BNT162b1 and the BNT162b2recipients, after the first and second dose and in both age groups, themajority of systemic events were mild or moderate in severity, and noGrade 4 systemic events were reported.

BNT162b1

In the younger age group, fatigue was the most frequently reportedsystemic event within 7 days after Dose 1 of BNT162b1, reported by 4(33.3%), 8 (66.7%), and 6 (50.0%) participants in the 10-μg, 20-μg, and30-μg dose groups, respectively (FIG. 88 ), compared to the placebogroup (2 [22.2%] participants). Headache (6 [50.0%] participants) andchills (7 [58.3%] participants) were reported in the 30-μg dose group,and ≤5 (8.1%) participant reported fever in each group through 30 μg. Inthe placebo group, headache (1 [11.1%]) participant) was reported andnone reported fever or chills. In the 100-μg dose group, higherfrequencies were reported compared to the 30-μg dose group: fatigue (10[83.3%] participants), headache (9 [75.0%] participants), chills (10[83.3%] participants), and fever (6 [50.0%] participants).

Within 7 days after Dose 2 of BNT162b1 in the younger age group,headache was the most frequently reported systemic event, reported byall 12 (100.0%) participants in the 30-μg dose group compared to none inthe placebo group, while fatigue and chills were reported by 10 (83.3%participants) and 8 (66.7%) participants in the 30-μg dose group,respectively. Fever was reported in 17% and 75% of participants in the20-μg and 30-μg dose groups, respectively. In the placebo group, 2(22.2%) participants reported fatigue, and none reported fever andchills.

In the older age group, fatigue was the most frequently reportedsystemic event within 7 days after Dose 1 of BNT162b1, with 7 (58.3%)and 6 (≤0.0)% of participants reporting fatigue in the 20-μg and 30-μgdose groups, respectively (FIG. 89 ), compared to 4 (44.4%) participantsin the placebo group. Headache (6 [50.0%] participants) and chills (2[16.7%] participants) were reported in the 30-μg dose group, and fever(3 [25.0%] participants) was reported only in the 30-μg dose group. Inthe placebo group, chills (2 [22.2%] participants) was reported and nonereported headache or fever. One participant each reported severe musclepain (20-μg dose group) and severe fatigue (30-μg dose group) (theformer was pain related to onset of herpes zoster).

Within 7 days after Dose 2 of BNT162b1 in the older age group, headachewas the most frequent systemic event reported in both the 20-μg and30-μg dose groups (9 [75.0%] participants each) compared to the placebogroup (1 [11.1%] participant). Chills was reported in 7 (58.3%) and 4(33.3%) participants at the 20-μg and 30-μg dose groups, respectively.Fever was reported in 6 (50.0%) participants in the 20-μg dose group andin 4 (33.3%) participants in the 30-μg dose group, with 1 participantreporting fever >38.9° C. to 40.0° C. In the placebo group, fatigue (2[22.2%] participants) was reported and none reported fever and chills.

After the first and second dose and in both age groups, the majority ofsystemic events were mild or moderate in severity, and no Grade 4systemic events were reported. In the older age group, prompted systemicevents after each dose were milder and less frequent than those observedin the younger age group.

Systemic events had the highest frequency and/or severity with the100-μg dose group after Dose 1. Use of antipyretic/pain medication alsoincreased with increasing dose level and number of doses in both agegroups. For these reasons, the IRC decided that the younger age groupparticipants should not receive a second dose of 100 μg of BNT162b1.

In the younger age group, median onset day for most systemic eventsafter either dose of BNT162b1 across doses 10 μg to 30 μg and after Dose1 of BNT162b1 100 μg was between Day 1.0 and Day 2.0. Most systemicevents generally resolved with median durations between 1.0 to 2.0 days.For fatigue, median duration after Dose 1 was 4.0 days in the 10-μg dosegroup compared with 2.0 days in the 30-μg dose group. In the older agegroup, median onset day for most systemic events after either dose ofBNT162b1, and across any dose group, was between Day 1.0 and Day 3.5.Most systemic events generally resolved with median durations between1.0 to 3.0 days.

BNT162b2

In the younger age group, headache (4 [33.3%] to 6 [50.0%] participants)and fatigue (3 [25.0%] to 5 [41.7%] participants) were the mostfrequently reported systemic events within 7 days after Dose 1 ofBNT162b2 compared to the placebo group (3 [33.3%] participants each)(FIG. 90 ). Fever (2 [16.7%] participants) and chills (4 [33.3%]participants) were reported only in the 30-μg dose group. Oneparticipant in the 30-μg group with a prior history of migraine reporteda severe migraine headache on Day 7 after Dose 1.

Within 7 days after Dose 2 of BNT162b2 in the younger age group, fatiguewas the most frequently reported systemic event in the 20-μg and 30-μgdose groups (7 [58.3%] and 9 [75.0%] participants, respectively)compared to the placebo group (5 [55.6%] participants). Headache (8[66.7%] participants), chills (7 [58.3%] participants), and muscle pain(7 [58.3%] participants), and fever (2 [16.7%] participants) werereported in the 30-μg dose group. Of these events, fatigue (5 [55.6%]participants), headache (1 [11.1%] participant), and chills (1 [11.1%]participant) were reported in the placebo group, and none were reportedfor muscle pain.

In the older age group, the most frequently reported systemic eventwithin 7 days after Dose 1 of BNT162b2 was fatigue in the 20-μg and at30-μg dose groups (4 [33.3%] and 3 [25.0%] participants, respectively)compared to the placebo group (2 [22.2%] participants) (FIG. 91 ).Headache (3 [25.0%] participants), chills (2 [16.7%] participants), andmuscle pain (1 [8.3%] participant) were maximal in the 20-μg dose group.Of these events, only headache (1 [11.1%] participant) and muscle pain(2 [22.2%] participants) were reported in the placebo group. Fever wasnot reported. Within 7 days after Dose 2 of BNT162b2 in the older agegroup, fatigue remained the most frequent systemic event in the 20-μgand 30-μg dose groups (6 [50.0%] and 5 [41.7%] participants,respectively), compared to the placebo group (1 [11.1%] participant).Headache was reported in the 20-μg and 30-μg dose groups (4 [33.3%] and3 [25.0%] participants, respectively), while muscle pain and chills werereported in the 30-μg dose group (3 [25.0%] and 2 [16.7%] participants,respectively). Fever (1 [8.3%] participant) was reported in the 30-μgdose group. Of these events, headache and muscle pain were reported inthe placebo group (1 [11.1%] participant each).

After the first and second dose and in both age groups, the majority ofsystemic events were mild or moderate in severity, and no Grade 4systemic events were reported. In the younger age group, median onsetday for most systemic events after either dose of BNT162b2, and acrossany dose group, was between Day 1.0 and Day 4.0. Most systemic eventsgenerally resolved with median durations between 1.0 to 2.5 days. In theolder age group, median onset day for any systemic event after eitherdose of BNT162b2, and across any dose level, was between Day 1.5 and Day2.0, except for systemic events in the 10-μg dose group after Dose 1,which had a median onset day of Day 5.5. Most systemic events generallyresolved with median durations between 1.0 to 3.0 days.

Adverse Events—Phase 1 Summary of Adverse Events—Phase 1

All AEs from Dose 1 through the data cutoff date of 24 Aug. 2020 wereincluded in the summary for all dose levels for each vaccine candidateand age group other than BNT162b1 100-μg group for which AEs from Dose 1to before Dose 2 were summarized.

Overall, fewer participants reported at least 1 AE after Dose 1 in theolder BNT162b2 group (8.3% to 25.0%) compared to the younger (41.7% to50.0%) and older (25.0% to 58.3%) BNT162b1 groups and the youngerBNT162b2 group (33.3% to 41.7%).

BNT162b1

In the younger age group, 5 (41.7%) to 6 (50%) participants reported atleast 1 AE after Dose 1 of BNT162b1 up to 30 μg, compared to 2 (22.2%)participants in the placebo group. Related AEs increased with increasingBNT162b1 dose level (25.0% to 50.0%); six (50%) participants reported atleast 1 related AE in the 30-μg dose group. One (8.3%) participantreported a severe AE (pyrexia) in the 30-μg dose group.

In the 100-μg dose group, 8 (66.7%) participants reported at least 1 AEafter Dose 1 to before Dose 2 of BNT162b1, compared to 1 (33.3%)participant in the placebo group. Six (50.0%) participants had at least1 related AE, and 1 (8.3%) participant reported a severe AE (sleepdisorder).

In the older group, 3 (25.0%) participants (30-μg dose group) and 7(58.3%) participants each (10-μg and 20-μg dose groups) reported atleast 1 AE after Dose 1 of BNT162b1, compared to 4 (44.4%) participantsin the placebo group. Two (16.7%) to 4 (33.3%) participants reported atleast 1 related AE, with the highest frequency in the 20-μg dose group.One participant each reported a severe AE in the 20-μg (herpes zoster)and 30-μg (fatigue) dose groups.

No SAEs, AEs leading to withdrawals, or deaths were reported in eitherage group.

BNT162b2

In the younger age group, 4 (33.3%) to 5 (41.7%) participants reportedat least 1 AE after Dose 1 of BNT162b2, compared to 2 (22.2%)participants in the placebo group. Two (16.7%) to 4 (33.3%) participantsreported at least 1 related AE, with the highest frequency in the 20-μgdose group. One participant reported a severe AE (migraine) in the 30-μgdose group.

In the older group, 1 (8.3%) to 3 (25.0%) participants reported at least1 AE after Dose 1 of BNT162b2, compared to 2 (22.2%) participants in theplacebo group. Only 1 (8.3%) participant reported at least 1 related AE(20-μg dose group). One participant each reported a severe AE in the30-μg dose group (muscle spasms) and placebo group (radiculopathy).

No SAEs, AEs leading to withdrawals, or deaths were reported in eitherage group.

Analysis of Adverse Events—Phase 1 Adverse Events by System Organ Classand Preferred Term—Phase 1

AE by SOC and PT summaries in this section included AEs from Dose 1 to 1month after Dose 2 for all groups other than BNT162b1 100-ug group forwhich AEs from Dose 1 to 3 weeks after Dose 1 or from Dose 1 to beforeDose 2 were summarized.

General disorders and administration site conditions was the mostcommonly reported SOC in the older BNT162b1 group and the youngerBNT162b2 group. The most commonly reported SOC was gastrointestinaldisorders in the younger BNT162b1 group and nervous system disorders inthe older BNT162b2 group. Generally, most PTs were reported by 52participants per dose group.

BNT162b1

In the younger age group, from Dose 1 to 1 month after Dose 2 ofBNT162b1, gastrointestinal disorders was the most commonly reported SOC(2 [16.7%] participants each dose group) in the BNT162b1 groups up to 30μg. In the 20-μg dose group only, paraesthesia (3 [25.0%]) was the mostcommon AE by PT. All other AEs were reported by ≤2 participants perdosegroup, including those in the placebo group. In the 100-μg dose group,from Dose 1 to 3 weeks after Dose 1 of BNT162b1, psychiatric disorderswas the most commonly reported SOC (3 [25.0%] participants), and sleepdisorder (3 [25%] participants) was the most common AE by PT. All otherAEs were reported by ≤2 participants, including those in the placebogroup.

In the older age group, from Dose 1 to 1 month after Dose 2 of BNT162b1,general disorders and administration site conditions was the mostcommonly reported SOC in the BNT162b1 groups, reported in a total of 6participants: 1 (8.3%) participant in the 10-μg dose group, 2 (16.7%)participants in the 20-μg dose group, and 3 (25.0%) participants in the30-μg dose group. Any AEs by PT were reported by no more than 1participant per dose group.

BNT162b2

In the younger age group, general disorders and administration siteconditions was the most commonly reported SOC. These events includedinjection site pain and injection site erythema. Any AEs by PT werereported by no more than 1 participant per dose group.

In the older age group, nervous system disorders was the most commonlyreported SOC, reported in 1 participant each in the 30-μg group(sciatica) and the placebo group (radiculopathy). Any AEs by PT werereported by no more than 1 participant per dose group.

Related Adverse Events—Phase 1

Overall, general disorders and administration site conditions was themost commonly reported SOC for the younger and older BNT162b1 groups andthe younger BNT162b2 group. In the older BNT162b2 group, nausea,reported in 1 (8.3%) participant, was the only related AE.

BNT162b1

In the younger age group, general disorders and administration siteconditions was the most commonly reported SOC (injection site pain,pyrexia, chills, fatigue, and injection site swelling). Two (16.7%)participants each in the 30-μg dose group reported related AEs oftachycardia and pyrexia. All other related AEs were reported by ≤2participants per dose group.

In the 100-μg BNT162b1 group, psychiatric disorders were the mostcommonly reported SOC. Three (25.0%) participants reported sleepdisorder as their psychiatric disorder. All other related AEs werereported by 52 participants each.

In the older age group, general disorders and administration siteconditions was the most commonly reported SOC (fatigue, injection sitebruising, injection site pain, and peripheral swelling). Any related AEsby PT were reported by no more than 1 participant per dose group.

BNT162b2

In the younger age group, general disorders and administration siteconditions was the most commonly reported SOC (injection site pain andinjection site erythema). Any related AEs by PT were reported by no morethan 1 participant per dose group, including those in the placebo group.

In the older age group, only 1 (8.3%) participant reported a related AEof nausea in the 20-μg dose group.

Immediate Adverse Events—Phase 1 BNT162b1

In the younger age group, 1 participant reported an immediate AE ofparaesthesia after Dose 1 of 20 μg BNT162b1. In the 100-μg group, noparticipants reported an immediate AE after Dose 1.

In the older age group, 1 participant reported an immediate AE of eyeparaesthesia after Dose 1 of 10 μg BNT162b1.

There were no participants in either age group who reported anyimmediate AEs after Dose 2 of BNT162b1.

BNT162b2

In the younger age group, after Dose 1 of BNT162b2, there were 3participants who reported an immediate AE: injection site erythema(10-μg dose group), ageusia (20-μg dose group), and injection site pain(30-μg dose group). After Dose 2 of BNT162b2, there was 1 participantwho reported an immediate AE of taste disorder (20-μg dose group).

There were no participants in the older age group who reported anyimmediate AE after any dose of BNT162b2.

Severe Adverse Events—Phase 1 BNT162b1

In the younger age group, there was 1 participant who reported a severeAE of pyrexia (102.4° F.) 2 days after Dose 2 (30-μg dose group) and 1participant who reported a severe AE of sleep disorder 1 day after Dose1 (100-μg dose group). Both AEs were determined by the investigator tobe related to study intervention.

In the older age group, 2 participants reported a severe AE: herpeszoster which occurred 2 days after Dose 1 (20-μg dose group, consideredunrelated to BNT162b1) and fatigue 1 day after Dose 2 (30-μg dose group,considered related to BNT162b1).

BNT162b2

In the younger age group, 1 participant with a history of migrainesreported a severe migraine 7 days after Dose 1 (30-μg dose group,considered unrelated). In the older age group, 2 participants reported asevere AE: muscle spasms 2 days after Dose 2 (30-μg dose group,considered unrelated to BNT162b2) and radiculopathy 3 days after Dose 1(placebo), considered unrelated to study intervention.

Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals,and Other Significant Adverse Events—Phase 1 Deaths—Phase 1

There were no Phase 1 participants who died through the data cutoff dateof 24 Aug. 2020 in this interim CSR.

Serious Adverse Events—Phase 1

There were no Phase 1 participants who reported any SAEs during theperiod covered in this interim CSR.

Safety-Related Participant Withdrawals—Phase 1

There were no Phase 1 participants with any AEs leading to withdrawalfrom the study through the data cutoff date of 24 Aug. 2020 in thisinterim CSR.

Other Significant Adverse Events—Phase 1

AEs of special interest were not defined for Phase 1 of this study.

Other Safety Assessments—Phase 1 Severe COVID-19 Illness—Phase 1

There were no COVID-19 cases reported in the Phase 1 participantsthrough the data cutoff date of 24 Aug. 2020.

Pregnancy—Phase 1

Pregnancy was not reported in any Phase 1 participants through the datacutoff date of 24 Aug. 2020.

Analysis and Discussion of Deaths, Serious Adverse Events,Safety-Related Participant Withdrawals, and Other Significant AdverseEvents—Phase 1

During the period covered in this interim CSR, there were no SAEs, AEsleading to withdrawals, or deaths reported in either age group.

Clinical Laboratory Evaluation—Phase 1

Overall, 1 to 3 days after Dose 1, there were transient decreases inlymphocytes (<0.8×LLN), which returned to normal by 6 to 8 days afterDose 1, in the younger and older BNT162b1 and BNT162b2 groups. Mostshifts were from normal or Grade 1 to Grade 1, 2, or 3 decrease inlymphocyte counts, which returned to normal by 6 to 8 days after Dose 1,and were observed in all age and dose groups. Shifts from normal toGrade 1 (younger BNT162b1 group) or Grade 2 (older BNT162b2 group)neutrophil decrease were also observed but were infrequent.

Overall, other clinical chemistry abnormalities reported or shifts oflaboratory results were infrequent. The incidence of decreasedlymphocyte counts was lower for BNT162b2 recipients compared withBNT162b1 recipients. None of the laboratory abnormalities wereassociated with clinical findings.

BNT162b1

In the younger age group, laboratory abnormalities of transientdecreases in lymphocytes (<0.8×LLN) were observed in 1 (8.3%), 4(33.3%), and 6 (54.5%) of participants 1 to 3 days after Dose 1 ofBNT162b1 10 μg, 20 μg, or 30 μg, respectively, which returned to normalby 6 to 8 days after Dose 1. A shift from normal to Grade 3 decrease inlymphocyte counts was observed in 1 participant each in the 10-μg and30-μg dose groups and 2 (16.7%) participants the 20-μg dose group. NoGrade 3 decrease in lymphocyte counts was observed by 6 to 8 days afterDose 1. After Dose 1, a shift from normal to Grade 2 neutrophil decreasewas observed in 1 (11.1%) participant in the placebo group, which wasnot observed by 19 to 23 days after Dose 1. At 6 to 8 days after Dose 2,a shift in neutrophil decrease was observed in 1 participant each in the10-μg dose group (Grade 1 to Grade 2) and in the 30-μg dose group(normal to Grade 2). Both participants had a shift to Grade 1 at theunplanned visit approximately 1 month after Dose 2.

In the 100-μg BNT162b1 group, laboratory abnormalities of transientdecreases in lymphocytes (<0.8×LLN) were observed in 9 (75.0%)participants 1 to 3 days after Dose 1, which returned to normal by 6 to8 days after Dose 1. A shift from normal to Grade 3 decrease inlymphocyte counts was observed in 4 (33.3%) participants 1 to 3 daysafter Dose 1, which returned to normal by 6 to 8 days after Dose 1. Ashift from normal to Grade 1 neutrophil decrease was observed in 3(25.0%) participants at 6 to 8 days after Dose 1, which returned tonormal by 19 to 23 days after Dose 1.

In the older age group, laboratory abnormalities of transient decreasesin lymphocytes (<0.8×LLN) were also observed in 1 (8.3%), 3 (25.0%), and2 (16.7%) participants 1 to 3 days after Dose 1 of BNT162b1 10 μg, 20μg, or 30 μg, respectively, which returned to normal by 6 to 8 daysafter Dose 1. At 1 to 3 days after Dose 1 of BNT162b1, shifts fromnormal to Grade 3 or Grade 4 decrease in lymphocyte counts were observedin 1 (8.3%) participant each in the 30-μg and 10-μg dose groups,respectively, and both returned to normal by 6 to 8 days after Dose 1.

Overall, other clinical chemistry abnormalities reported or shifts oflaboratory results were infrequent. None of the abnormalities wereassociated with clinical findings.

BNT162b2

In the younger age group, laboratory abnormalities of transientdecreases in lymphocytes (<0.8×LLN) were observed in 1 (8.3%)participant each 1 to 3 days after Dose 1 of BNT162b2 in the 20-μg and30-μg dose groups, which returned to normal by 6 to 8 days after Dose 1.At 1 to 3 days after Dose 1 of BNT162b2, shifts from normal to Grade 1decrease in lymphocyte counts were observed in 3 (25.0%), 2 (16.7%), and4 (33.3%) participants in the 10-μg, 20-μg, and 30-μg dose groups,respectively, and shifts from normal to Grade 2 decrease in lymphocytecounts were observed in 1 (8.3%) participant each in the 20-μg and 30-μgdose groups. By 6 to 8 days after Dose 1, no Grade 2 or Grade 3 decreasein lymphocyte counts were observed.

In the older age group, laboratory abnormalities of transient decreasesin lymphocytes (<0.8×LLN) were also observed in 1 (8.3%) participanteach 1 to 3 days after Dose 1 of BNT162b2 across all dose levels, whichreturned to normal by 6 to 8 days after Dose 1. A shift from normal toGrade 3 (10-μg dose group) and a Grade 1 to Grade 3 (30-μg dose group)decrease in lymphocyte counts was observed in 1 (8.3%) participant eachafter Dose 1. A shift from normal to Grade 2 neutrophil decrease wasobserved in 2 (16.7%) participants in the 20-μg dose group at 1 to 3days after Dose 1, and no shifts to Grade 2 were observed by 6 to 8 daysafter Dose 1. A shift from normal to Grade 2 neutrophil decrease wasobserved in 1 (8.3%) participant in the 10-μg dose group at 6 to 8 daysafter Dose 1. By 19 to 23 days after Dose 1, no shifts to Grade 2neutrophil decrease were observed for any dose group.

Overall, other clinical chemistry abnormalities reported or shifts oflaboratory results were infrequent. The incidence of decreasedlymphocyte counts was lower for BNT162b2 recipients compared withBNT162b1 recipients. None of the laboratory abnormalities wereassociated with clinical findings.

Physical Examination Findings—Phase 1

Overall, there were fewer abnormalities noted during physicalexaminations after BNT162b2 than after BNT162b1 in both age groups.Abnormalities were generally observed 1 to 3 days after Dose 1 and mostwere of the extremities, musculoskeletal system, or skin.

BNT162b1

In the younger age group, there were no abnormalities noted duringbaseline physical examinations. Overall, after randomization, mostabnormalities were observed 1 to 3 days after Dose 1 of 10 μg, 20 μg, or30 μg BNT162b1 (9 [20.0%] participants) and 6 to 8 days after Dose 2 (7[15.6%] participants). In the 30-μg dose group, a maximum of 6 (50.0%)participants had abnormalities 1 to 3 days after Dose 1, and mostabnormalities were of the extremities.

In the 100-μg dose group, only 1 (8.3%) participant had an abnormalityat baseline. From Dose 1 to 3 weeks after Dose 1, 9 (75.0%) participantshad abnormalities 1 to 3 days after BNT162b1, and most abnormalitieswere of the extremities.

In the older age group, there were 5 (11.1%) participants withabnormalities noted during baseline physical examinations, with ≤2participants in any dose group. Overall, after randomization, mostabnormalities were observed 1 to 3 days after Dose 1 of BNT162b1 (15[33.3%] participants). In the 20-μg and 30-μg dose groups, 6 (50.0%) and4 (33.3%) participants had abnormalities 1 to 3 days after Dose 1, andmost abnormalities involved either the musculoskeletal system orextremities.

There were no clinically important findings from physical examinations.

BNT162b2

In the younger age group, there were 5 (11.1%) participants withabnormalities noted during baseline physical examinations, with ≤2participants in any dose group. Overall, after randomization, mostabnormalities were observed 1 to 3 days after Dose 1 of 10 μg, 20 μg, or30 μg BNT162b2 (5 [11.1%] participants) and 6 to 8 days after Dose 2 (4[8.9%] participants), with most being abnormalities of the extremitiesor skin.

In the older age group, there was 1 (8.3%) participant in the 30-μg dosegroup with an abnormality noted during the baseline physicalexamination. After randomization, ≤2 participants in any dose groupoverall had an abnormality in physical examination during any visitwindow.

There were no clinically important findings from physical examinationsat baseline.

Phase 1 Summary of Safety Results Evaluating BNT162b1 and BNT162b2

Overall, reactogenicity events were well tolerated and short-lived(median durations 1.0 to 4.0 days). All participants returned to receivetheir second dose. All AEs as a result of reactogenicity events resolvedwithout sequelae.

For local reactions in both age groups, pain at the injection site(58.3% to 100.0%), redness (0% to 16.7%), and swelling (0% to 25.0%)were reported for BNT162b1 recipients, which were more frequent than forBNT162b2 recipients: pain at the injection site (33.3% to 91.7%),redness (0% to 8.3%), and swelling (0% to 16.7%). In general,frequencies of local reactions were observed to be higher with increaseddose level.

The frequency of local reactions was lower in the older age groupcompared to the younger age group. The frequency of pain at theinjection site, the most frequently reported local reaction, was lowerin the older age groups after 30 μg BNT162b1 (91.7% and 75.0%) and after30 μg of BNT162b2 (75.0% and 66.7% for Dose 1 and Dose 2, respectively),compared to the younger age groups after 30 μg of BNT162b1 (100.0% forboth Dose 1 and Dose 2) and 30 μg of BNT162b2 (91.7% and 83.3% for Dose1 and Dose 2, respectively).

BNT162b2 recipients in the older age group reported lower frequencies oflocal reactions compared with BNT162b1 recipients in the older agegroup. In the older 30-μg BNT162b2 group, pain at the injection site waslower after Dose 1 (75.0%) and Dose 2 (66.7%) than in the older 30-μgBNT162b1 group after Dose 1 (91.7%) and Dose 2 (75.0%).

Common systemic events in both age groups after either Dose 1 or Dose 2included fatigue (16.7% to 83.3%), headache (25.0% to 100%), chills(8.3% to 66.7%), fever (0% to 75.0%), and muscle pain (8.3% to 75.0%)for BNT162b1 recipients up to 30 μg, which were more frequent thanBNT162b2 recipients up to 30 μg: fatigue (8.3% to 75.0%), headache (0%to 66.7%), chills (0% to 58.3%), fever (0% to 16.7%), and muscle pain(0% to 58.3%). In general, frequencies of systemic events were observedto be higher with increased dose level.

The frequency of systemic events was lower in the older age groupcompared to the younger age group. The frequency of fatigue was lower inthe older age groups after 30 μg of BNT162b1 (50.0% and 66.7%) and after30 μg of BNT162b2 (25.0% and 41.7% for Dose 1 and Dose 2, respectively),compared to the younger age groups after 30 μg of BNT162b1 (50.0% and83.3%) and after 30 μg of BNT162b2 (41.7% and 75.0%) for Dose 1 and Dose2, respectively.

BNT162b2 recipients in the older age group reported lower frequencies ofsystemic events compared with BNT162b1 recipients in the older agegroup. The frequency of fatigue was lower in the older 30-μg BNT162b2group (25.0% and 41.7% for Dose 1 and Dose 2, respectively) than in theolder 30-μg BNT162b1 group (50.0% and 66.7% for Dose 1 and Dose 2,respectively).

Most AEs were mild or moderate in severity. Most related AEs weresimilar to the solicited reactogenicity events reported in the e-diary.Few severe AE were reported but were considered not related to studyintervention.

There were no SAEs, deaths, or discontinuations because of AEs.

Transient decrease in lymphocytes were observed in all age and dosegroups 1 to 3 days after Dose 1, which resolved by 6 to 8 days afterDose 1.

There were no clinically important findings from physical examinations.

BNT162b2 demonstrated a favorable reactogenicity and safety profilecompared with BNT162b1, contributing to the selection of BNT162b2 forPhase 2/3 development.

Phase 1 Safety Conclusions

All doses tested for BNT162b1 and BNT162b2 (10 μg, 20 μg, and 30 μg)were safe and well tolerated except for BNT162b1 at 100 μg, which wasdiscontinued after the first dose due to the reactogenicity profile.

Reactogenicity was generally higher after Dose 2 than Dose 1.

The frequency of local and systemic reactogenicity was generally lowerfor BNT162b2 compared to BNT162b1 especially after the second dose.

Reactogenicity events after each dose for both BNT162b1 and BNT162b2 inolder adults were milder and less frequent than those observed inyounger adults. The majority of reactogenicity events were mild ormoderate in severity.

Most AEs were mild or moderate. There were no SAEs or discontinuationsbecause of AEs.

Overall, fewer AEs were experienced by participants who receivedBNT162b2 compared with those who received BNT162b1, with the leastnumber of participants experiencing AEs in the BNT162b2 older age group.Few severe AEs in the older age group after BNT162b2 were observed, andall were considered unrelated to study intervention.

Clinical laboratory evaluations showed a transient decrease inlymphocytes that was observed in all age and dose groups after Dose 1,which resolved within a few days, were not associated with any otherclinical sequelae, and were not considered clinically relevant.

BNT162b2 at 30 μg was selected to proceed into the Phase 2/3 portion ofthe study because this dose and construct provided the optimumcombination of a favorable reactogenicity profile and a robust immuneresponse.

Phase 2

Safety data are available up to the data cutoff date (2 Sep. 2020) andare summarized up to the data cutoff date for the 360 participants inPhase 2. All participants in Phase 2 used an e-diary for reporting localreactions and systemic events.

Local Reactions—Phase 2

After the first and second dose of BNT162b2 and in both age groups, themajority of local reactions were mild or moderate in severity, and noGrade 4 (potentially life-threatening) local reactions were reported.

In the BNT162b2 group, pain at the injection site was reported morefrequently in the younger age group (FIG. 92 ) than in the older agegroup (FIG. 93 ), and frequency was similar after Dose 1 compared withDose 2 of BNT162b2 in the younger age group (85.2% vs. 80.2%,respectively) and in the older age group (70.7% vs. 72.5%,respectively). In the placebo group, pain at the injection site wasreported at similar frequencies (7.8% to 10.2%) in the younger and olderage groups after Dose 1 and Dose 2.

In the BNT162b2 group, redness and swelling were similar in the youngerand older age group after Dose 1. After Dose 2, the frequency of rednessand swelling was slightly higher in the older age group (7.7% and 12.1%,respectively) than in the younger age group (3.5% and 3.5%,respectively). In the placebo group, only 1 participant in the older agegroup reported redness after Dose 1, and no swelling was reported.

One participant in the BNT162b2 group (older age group) reported severeinjection site pain after Dose 1, and 1 participant in the younger agegroup reported severe injection site pain after Dose 2. One participantin the BNT162b2 group (older age group) reported severe redness afterDose 2.

Overall, across age groups, pain at the injection site was the mostfrequent local reaction and did not increase after Dose 2, and rednessand swelling were generally similar in frequency after Dose 1 and Dose2.

Across age groups, local reactions for the BNT162b2 group after eitherdose had a median onset day between Day 1.0 and Day 3.0 (Day 1.0 was theday of vaccination), and ranges were generally similar in the youngerand older age groups. Across age groups, after either dose of BNT162b2,local reactions resolved after a median duration of 1.0 to 3.0 days,which was generally similar in the younger and older age groups.

Systemic Events—Phase 2

In the BNT162b2 group, systemic events were generally reported morefrequently and were of higher severity in the younger group (FIG. 94 )compared with the older group (FIG. 95 ), with frequencies and severityincreasing with number of doses (Dose 1 vs Dose 2). Vomiting anddiarrhea were exceptions with vomiting infrequent and similar in bothage groups and vomiting and diarrhea similar after each dose.Frequencies of systemic events in the younger and older BNT162b2 groups(Dose 1 vs Dose 2) are listed below:

fatigue: younger group (50.0% vs 59.3%) compared to older group (35.9%vs 52.7%)headache: younger group (31.8% vs 51.2%) compared to older group (27.2%vs 36.3%)muscle pain: younger group (23.9% vs 45.3%) compared to older group(14.1% vs 28.6%)chills: younger group (9.1% vs 40.7%) compared to older group (7.6% vs20.9%)joint pain: younger group (9.1% vs 17.4%) compared to older group (4.3%vs 16.5%)fever: younger group (3.4% vs 17.4%) compared to older group (0.0% vs11.0%).vomiting: similar in both age groups and after either dose.diarrhea: reported less frequently in the older group and was similarafter each dose. Systemic events were generally reported less frequentlyin the placebo group than in the BNT162b2 group, for both age groups anddoses, with some exceptions. In the younger age group, fever, headache,chills, vomiting, and diarrhea after Dose 1, and vomiting after Dose 2were reported at similar frequencies in both the placebo and BNT162b2groups (FIG. 94 ). In the older age group, vomiting, diarrhea, musclepain, and joint pain after Dose 1, and vomiting and diarrhea after Dose2 were reported at similar frequencies in the placebo and BNT162b2groups (FIG. 95 ).

Use of antipyretic/pain medication was slightly less frequent in theolder age group after both doses but increased in both age groupsoverall after Dose 2 as compared with after Dose 1. Use ofantipyretic/pain medication was less frequent in the placebo group thanin the BNT162b2 group.

After the first and second dose and in both age groups, the majority ofsystemic events were mild or moderate in severity, and no Grade 4(potentially life-threatening) systemic events were reported. Across agegroups, severe systemic events were only reported after Dose 2 ofBNT162b2 overall and included fever (1.1%), fatigue (4.0%), headache(2.8%), chills (2.3%), and muscle pain (1.7%).

Across age groups, systemic events after both doses of BNT162b2 had amedian onset day between Day 2.0 to Day 3.0 (Day 1.0 was the day ofvaccination), and ranges were similar in the younger and older agegroups. Across age groups, systemic events for this group after eitherdose resolved with a median duration of 1 day, which was similar in theyounger and older age groups. There was no clear difference in thedurations of systemic events that occurred after Dose 1 compared tothose that occurred after Dose 2.

Adverse Events—Phase 2 Summary of Adverse Events—Phase 2

The number of participants who reported at least 1 AE was similar in theBNT162b2 group compared with the placebo group, which was generallysimilar in the 2 vaccine groups in the younger and older age groups(Table 10 and Table 11, respectively).

Two severe events were reported for 2 participants in the BNT162b2younger age group: myalgia (AE) and gastric adenocarcinoma (SAE) The SAEof gastric adenocarcinoma occurred 23 days after receiving Dose 1. Bothevents were assessed by the investigator as not related to studyintervention.

From 7 days after Dose 2 to the data cutoff date (2 Sep. 2020), noadditional participants reported any AE.

TABLE 10 Number (%) of Subjects Reporting at Least 1 Adverse Event FromDose 1 to 7 Days After Dose 2, by Age Group - Phase 2 - SafetyPopulation Age Group: 18-55 Years Vaccine Group (as Administered)BNT162b2 (30 μg) Placebo (N^(a) = 88) (N^(a) = 90) Adverse Event n^(b)(%) n^(b) (%) Any event 8 (9.1) 10 (11.1) Related^(c) 3 (3.4) 6 (6.7)Severe 2 (2.3) 0 Life-threatening 0 0 Any serious adverse event 1 (1.1)0 Related^(c) 0 0 Severe 1 (1.1) 0 Life-threatening 0 0 Any adverseevent leading 1 (1.1) 0 to withdrawal Related^(c) 0 0 Severe 1 (1.1) 0Life-threatening 0 0 Death 0 0 ^(a)N = number of subjects in thespecified group. This value is the denominator for the percentagecalculations. ^(b)n = Number of subjects reporting at least 1 occurrenceof the specified adverse event category. For “any event”, n = the numberof subjects reporting at least 1 occurrence of any adverse event.^(c)Assessed by the investigator as related to investigational product.

TABLE 11 Number (%) of Subjects Reporting at Least 1 Adverse Event FromDose 1 to 7 Days After Dose 2, by Age Group - Phase 2 - SafetyPopulation Age Group: 56-85 Years Vaccine Group (as Administered)BNT162b2 (30 μg) Placebo (N^(a) = 92) (N^(a) = 90) Adverse Event n^(b)(%) n^(b) (%) Any event 4 (4.3) 8 (8.9) Related^(c) 2 (2.2) 2 (2.2)Severe 0 0 Life-threatening 0 0 Any serious adverse event 0 0Related^(c) 0 0 Severe 0 0 Life-threatening 0 0 Any adverse eventleading 0 0 to withdrawal Related^(c) 0 0 Severe 0 0 Life-threatening 00 Death 0 0 ^(a)N = number of subjects in the specified group. Thisvalue is the denominator for the percentage calculations. ^(b)n = Numberof subjects reporting at least 1 occurrence of the specified adverseevent category. For “any event”, n = the number of subjects reporting atleast 1 occurrence of any adverse event. ^(c)Assessed by theinvestigator as related to investigational product.

Analysis of Adverse Events—Phase 2 Adverse Events by System Organ Classand Preferred Term—Phase 2

Table 12 presents the number of participants who reported at least 1 AEfrom Dose 1 to 7 days after Dose 2 by SOC and PT.

The number of participants who reported at least 1 AE was similar in theBNT162b2 group compared to the placebo group from Dose 1 to 7 days afterDose 2.

In the younger age group, 8 (9.1%) and 10 (11.1%) participants reportedat least 1 AE in the BNT162b2 group and the placebo group, respectively.In the older age group, 4 (4.3%) and 8 (8.9%) participants reported atleast 1 AE in the BNT162b2 group and the placebo group, respectively.

Overall, most AEs reported up to 7 days after Dose 2 were in the SOCs ofgastrointestinal disorders (3 [1.7%] in the BNT162b2 group and 2 [1.1%]in the placebo group), general disorders and administration siteconditions (3 [1.7%] in the BNT162b2 group and 7 [3.9%] in the placebogroup), and musculoskeletal and connective tissue disorders (3 [1.7%] inthe BNT162b2 group and 1 [0.6%] in the placebo group).

The most frequently reported AE by PT was injection site pain (3 [3.4%])in the younger BNT162b2 group, which all occurred on the day ofvaccination with Dose 1 during the reporting period for local reactions.Two events resolved within 3 days, and 1 event resolved 11 days later.All other AEs by PT were reported in 52 participants in each vaccinegroup.

One participant in the older BNT162b2 group had an AE of contusion inthe upper left arm deltoid region, which was assessed by theinvestigator as related to study intervention.

TABLE 12 Number (%) of Subjects Reporting at Least 1 Adverse Event FromDose 1 to 7 Days After Dose 2, by System Organ Class and PreferredTerm - Phase 2 - Safety Population Vaccine Group (as Administered)BNT162b2 (30 μg) Placebo 18-55 Years 56-85 Years 18-85 Years 18-85 YearsSystem Organ Class (N^(a) = 88) (N^(a) = 92) (N^(a) = 180) (N^(a) = 180)Preferred Term n^(b) (%) (95% CI^(c)) n^(b) (%) (95% CI^(c)) n^(b) (%)(95% CI^(c)) n^(b) (%) (95% CI^(c)) Any event 8 (9.1)  (4.0, 17.1) 4(4.3)  (1.2, 10.8) 12 (6.7)   (3.5, 11.4) 18 (10.0)   (6.0, 15.3) BLOODAND LYMPHATIC 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0,2.0) SYSTEM DISORDERS Lymphadenopathy 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1(0.6) (0.0, 3.1) 0 (0.0, 2.0) GASTROINTESTINAL 1 (1.1) (0.0, 6.2) 2(2.2) (0.3. 7.6) 3 (1.7) (0.3, 4.8) 2 (1.1) (0.1, 4.0) DISORDERSDiarrhoea 1 (1.1) (0.0, 6.2) 1 (1.1) (0.0, 5.9) 2 (1.1) (0.1, 4.0) 1(0.6) (0.0, 3.1) Odynophagia 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6)(0.0, 3.1) 0 (0.0, 2.0) Tongue discomfort 0 (0.0, 4.1) 0 (0.0, 3.9) 0(0.0, 2.0) 1 (0.6) (0.0, 3.1) GENERAL DISORDERS 3 (3.4) (0.7, 9.6) 0(0.0, 3.9) 3 (1.7) (0.3, 4.8) 7 (3.9) (1.6, 7.8) AND ADMINISTRATION SITECONDITIONS Injection site erythema 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1(0.6) (0.0, 3.1) 2 (1.1) (0.1, 4.0) Injection site pain 3 (3.4) (0.7,9.6) 0 (0.0, 3.9) 3 (1.7) (0.3, 4.8) 0 (0.0, 2.0) Fatigue 0 (0.0, 4.1) 0(0.0, 3.9) 0 (0.0, 2.0) 2 (1.1) (0.1, 4.0) Chills 0 (0.0, 4.1) 0 (0.0,3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Injection site discolouration 0(0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Injection siteswelling 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1)INFECTIONS AND 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1)INFESTATIONS Vulvovaginal mycotic 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0)1 (0.6) (0.0, 3.1) infection INJURY, POISONING 0 (0.0, 4.1) 1 (1.1)(0.0, 5.9) 1 (0.6) (0.0, 3.1) 3 (1.7) (0.3, 4.8) AND PROCEDURALCOMPLICATIONS Contusion 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0,3.1) 1 (0.6) (0.0, 3.1) Fall 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) Muscle rupture 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) Tendon rupture 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) INVESTIGATIONS 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) White blood cell count 0 (0.0, 4.1) 0 (0.0, 3.9) 0(0.0, 2.0) 1 (0.6) (0.0, 3.1) increased MUSCULOSKELETAL AND 2 (2.3)(0.3, 8.0) 1 (1.1) (0.0, 5.9) 3 (1.7) (0.3, 4.8) 1 (0.6) (0.0, 3.1)CONNECTIVE TISSUE DISORDERS Myalgia 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1(0.6) (0.0, 3.1) 1 (0.6) (0.0, 3.1) Arthralgia 1 (1.1) (0.0, 6.2) 0(0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) Neck pain 0 (0.0, 4.1) 1(1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NEOPLASMS BENIGN, 1(1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) MALIGNANTAND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (1.1)(0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NERVOUS SYSTEM 0(0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) DISORDERSHeadache 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1)RESPIRATORY, THORACIC 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 2 (1.1)(0.1, 4.0) AND MEDIASTINAL DISORDERS Oropharyngeal pain 0 (0.0, 4.1) 0(0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Productive cough 0 (0.0, 4.1)0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Rhinorrhoea 0 (0.0, 4.1) 0(0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) SKIN AND SUBCUTANEOUS 1 (1.1)(0.0, 6.2) 1 (1.1) (0.0, 5.9) 2 (1.1) (0.1, 4.0) 1 (0.6) (0.0, 3.1)TISSUE DISORDERS Dermatitis 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) Hangnail 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0,3.1) 0 (0.0, 2.0) Macule 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0,3.1) 0 (0.0, 2.0) Rash macular 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1(0.6) (0.0, 3.1) Note: MedDRA (v23.0) coding dictionary applied. ^(a)N =number of subjects in the specified group. This value is the denominatorfor the percentage calculations. ^(b)n = Number of subjects reporting atleast 1 occurrence of the specified event. For “any event”, n = numberof subjects reporting at least 1 occurrence of any event. ^(c)Exact2-sided CI based on the Clopper and Pearson method.

Related Adverse Events by System Organ Class and Preferred Term—Phase 2

The number of participants with AEs assessed by the investigator asrelated to study intervention from Dose 1 to 7 days after Dose 2 werelow in frequency and similar in the BNT162b2 group and placebo group.Within the BNT162b2 group, a similar proportion of participants in theyoung and old age groups reported related AEs. Mostinvestigator-assessed related AEs were reactogenicity events in the SOCof general disorders and administration site conditions, and they werereported by a similar proportion of participants in the BNT162b2 groupoverall compared with the placebo group, with injection site pain beingthe PT reported most frequently and exclusively in the BNT162b2 youngerage group.

Immediate Adverse Events—Phase 2

There were no immediate AEs after any dose of BNT162b2 30 μg or placebo.

Severe or Life-Threatening Adverse Events—Phase 2

Two participants (both in the BNT162b2 younger age group) reportedsevere events of myalgia (AE) and gastric adenocarcinoma (SAE). Theparticipant who reported myalgia had scapular muscle pain, which began 2days after Dose 2 and was ongoing at the time of the data cutoff. Bothevents were assessed by the investigator as not related to studyintervention.

Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals,and Other Significant Adverse Events—Phase 2 Deaths—Phase 2

There were no Phase 2 participants who died through the data cutoff dateof 2 Sep. 2020 in this interim CSR.

Serious Adverse Events—Phase 2

One participant had an SAE from Dose 1 to 7 days after Dose 2 (Table13). One participant, who was in the BNT162b2 younger age group, had anSAE of gastric adenocarcinoma 23 days after Dose 1, which was assessedby the investigator as not related to study intervention (Table 13). TheSAE was ongoing at the time of the data cutoff, and the participant waswithdrawn from the study because of the SAE.

From 7 days after Dose 2 to the data cutoff date (2 Sep. 2020), noadditional participants reported any SAE.

TABLE 13 Number (%) of Subjects Reporting at Least 1 Serious AdverseEvent From Dose 1 to 7 Days After Dose 2, by System Organ Class andPreferred Term - Phase 2 - Safety Population Vaccine Group (asAdministered) BNT162b2 (30 μg) Placebo 18-55 56-85 18-85 18-85 SystemOrgan Years Years Years Years Class (N^(a) = 88) (N^(a) = 92) (N^(a) =180) (N^(a) = 180) Preferred Term n^(b) (%) (95% CI^(c)) n^(b) (%) (95%CI^(c)) n^(b) (%) (95% CI^(c)) n^(b) (%) (95% CI^(c)) Any event 1 (1.1)(0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NEOPLASMSBENIGN, 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0)MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma 1 (1.1)(0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) gastric Note:MedDRA (v23.0) coding dictionary applied. ^(a)N = number of subjects inthe specified group. This value is the denominator for the percentagecalculations. ^(b)n = Number of subjects reporting at least 1 occurrenceof the specified adverse event. For “any event”, n = number of subjectsreporting at least 1 occurrence of any adverse event. ^(c)Exact 2-sidedCI based on the Clopper and Pearson method.

Safety-Related Participant Withdrawals—Phase 2

The participant in the BNT162b2 younger age group who reported an SAE ofgastric adenocarcinoma was discontinued from the study on Day 23 afterDose 1 of BNT162b2.

Narratives of Safety-Related Participant Withdrawals—Phase 2

A narrative for the Phase 2 participant who was withdrawn from the studybecause of an SAE through the data cutoff date (2 Sep. 2020) wasprovided.

Other Significant Adverse Events—Phase 2

AEs of special interest were not defined for Phase 2 of this study;however, targeted medical events were monitored throughout the study.

Analysis and Discussion of Deaths, Serious Adverse Events,Safety-Related Participant Withdrawals, and Other Significant AdverseEvents—Phase 2

Up to the data cutoff date of 2 Sep. 2020, there was 1 participant inthe younger age group (BNT162b2 group) withdrawn from the study becauseof an SAE of gastric adenocarcinoma, which was assessed by theinvestigator as not related to study intervention.

Phase 2 Safety Conclusions

Across age groups, local reactions were generally similar in frequencyafter each dose, and systemic events generally increased in frequencyand severity after Dose 2 compared to Dose 1. Local and systemicreactogenicity events were well-tolerated and short-lived.

Reactogenicity events after each dose of BNT162b2 in older adults weregenerally milder and less frequent than those observed in youngeradults. The majority of reactogenicity events were mild or moderate inseverity. No Grade 4 events were reported.

AEs in participants were low in frequency, and most AEs were mild ormoderate in severity. There were no SAEs or discontinuations because ofAEs that were assessed as related by the investigator.

The reactogenicity and AE profile after BNT162b2 30 μg evaluated in 360participants was consistent with the safety profile observed afterBNT162b2 30 μg in Phase 1. BNT162b2 at 30 μg was safe and well toleratedup to 7 days after Dose 2.

Phase 2/3

In this interim CSR, safety results for Phase 3 included 36,855 olderadolescent and adult participants (16 to 91 years of age) up through thesafety data cutoff date of 6 Oct. 2020. AE summaries included any AEreported, regardless of whether participants completed the visit at 1month after Dose 2. The first 6610 adult participants (18 to 85 years ofage, which included the 360 participants in Phase 2) used an e-diary forreporting local reactions and systemic events and had safety datasummarized through at least 1 month after Dose 2.

During the Phase 2/3 portion of the study, a stopping rule for thetheoretical concern of vaccine enhanced disease was to be triggered ifthe 1-sided probability of observing the same or more extreme adversesevere case split was 5% or less, given the same true incidence forvaccine and placebo recipients, and alert criteria were to be triggeredif this probability was less than 11%. It is also noted that, with^(˜)18,000 per arm, the study has >83% probability of detecting at least1 adverse event.

Local Reactions—Phase 2/3

In the BNT162b2 group, pain at the injection site was reported morefrequently in the younger age group (FIG. 96 ) than in the older agegroup (FIG. 97 ), and frequency was similar after Dose 1 compared withDose 2 of BNT162b2 in the younger age group (85.3% vs. 79.5%,respectively) and in the older age group (71.7% vs. 66.6%,respectively). In the placebo group, pain at the injection site afterDoses 1 and 2 was reported at slightly higher frequencies in the youngerage group (13.8% and 11.9%, respectively) than in the older age group(8.8% and 7.7%, respectively).

In the BNT162b2 group, frequencies of redness and swelling were similarin the younger and older age group after Doses 1 and 2. Frequencies ofredness were similar after Dose 1 compared with Dose 2 of BNT162b2 inthe younger age group (4.3% vs 5.4%, respectively) and in the older agegroup (4.5% vs 6.6%, respectively). Frequencies of swelling were similarafter Dose 1 compared with Dose 2 of BNT162b2 in the younger age group(5.5% vs 5.9%, respectively) and in the older age group (6.5% vs 7.0%,respectively). In the placebo group, redness and swelling were reportedinfrequently in the younger (≤0.8%) and older (≤1.3%) age groups afterDoses 1 and 2. Overall, across age groups, pain at the injection sitedid not increase after Dose 2, and redness and swelling were generallysimilar in frequency after Dose 1 and Dose 2. Severe local reactions(≤0.8%) were reported infrequently in the BNT162b2 group after eitherdose overall but occurred more frequently in the younger group. Afterthe first and second dose and in both age groups, the majority of localreactions were mild or moderate in severity, and no Grade 4 localreactions were reported.

Subgroup Analyses

No clinically meaningful differences in local reactions were observed bycountry, sex, race, or ethnicity.

Across age groups, local reactions for the BNT162b2 group after eitherdose had a median onset day between Day 1.0 and Day 3.0 (Day 1.0 was theday of vaccination), and ranges were similar in the younger and olderage groups. Across age groups, local reactions for this group aftereither dose resolved with median durations between 1.0 to 2.0 days,which were similar in the younger and older age groups.

Systemic Events—Phase 2/3

Systemic events were generally increased in frequency and severity inthe younger group (FIG. 98 ) compared with the older group (FIG. 99 ),with frequencies and severity increasing with number of doses (Dose 1 vsDose 2) Vomiting and diarrhea were exceptions with vomiting reportedsimilarly infrequently in both age groups and both vomiting and diarrheasimilar after each dose. Frequencies of systemic events in the youngerand older BNT162b2 groups (Dose 1 vs Dose 2) are listed below:

fatigue: younger group (49.0% vs 61.6%) compared to older group (34.3%vs 51.2%)headache: younger group (42.9% vs 53.1%) compared to older group (25.4%vs 39.5%)muscle pain: younger group (22.0% vs. 38.6%) compared to older group(14.0% vs 28.5%)chills: younger group (14.4% vs 36.5%) compared to older group (6.2% vs22.8%)joint pain: younger group (10.9% vs 22.4%) compared to older group (8.3%vs 18.9%)fever: younger group (3.7% vs 16.6%) compared to older group (1.4% vs11.5%).vomiting: similar in both age groups and after either dose.diarrhea: reported less frequently in the older group and was similarafter each dose. Systemic events were generally reported less frequentlyin the placebo group than in the BNT162b2 group, for both age groups anddoses, with some exceptions. In the younger age group, fever and jointpain (after Dose 1) and vomiting and diarrhea (after Dose 1 and Dose 2)were reported at similar frequencies in the placebo group and theBNT162b2 group (FIG. 98 ). In the older age group, fever and joint pain(after Dose 1) and vomiting and diarrhea (after Dose 1 and Dose 2) werereported at similar frequencies in the placebo group and the BNT162b2group (FIG. 99 ).

Use of antipyretic/pain medication was slightly less frequent in theolder age group (20.1% to 37.4%) than in the younger age group (28.1% to45.8%) after both doses, and medication use increased in both age groupsafter Dose 2 as compared with after Dose 1. Use of antipyretic/painmedication was less frequent in the placebo group than in the BNT162b2group and was similar after Dose 1 and Dose 2 in the younger and olderplacebo groups (9.8% to 13.7%).

Severe systemic events across age groups after Dose 1 of BNT162b2 weregenerally lower in frequency than after Dose 2: fever (0.1% vs 0.8%),fatigue (0.8% vs 3.7%), headache (0.5% vs 1.9%), chills (0.2% vs 1.7%),muscle pain (0.3% vs. 1.6%), and joint pain (0.1% vs 0.6%). Diarrhea andvomiting frequencies were generally similar.

In the placebo group, severe fever was reported at a similar frequency(0.1%) after Dose 1 and Dose 2. One participant in the younger BNT162b2group reported fever of 41.2° C. only on Day 2 after Dose 2 and wasnonfebrile for all other days of the reporting period.

After the first and second dose and in both age groups, the majority ofsystemic events were mild or moderate in severity, and no Grade 4(potentially life-threatening) systemic events were reported other thanfever occurring only 1 day in 1 participant (41.2° C.) in the BNT162b2group.

Subgroup Analyses

No clinically meaningful differences in systemic events were observed bycountry, ethnicity, sex, or race.

Across age groups, median onset day for most systemic events aftereither dose of BNT162b2 was Day 2.0 (Day 1.0 was the day ofvaccination), and ranges were similar in the younger and older agegroups. Across age groups, all systemic events resolved with medianduration of 1.0 day, which was similar in the younger and older agegroups.

Adverse Events—Phase 2/3

In this interim CSR, the first 6610 adult participants (which includedthe 360 participants in Phase 2) had safety data summarized through atleast 1 month after Dose 2. AE summaries for all 36,855 participants upto the cutoff date (6 Oct. 2020) included any event reported, regardlessof whether participants completed the visit at 1 month after Dose 2. Atthe time of the data cutoff date, there was a small percentage (≤0.7%)of participants with at least 1 uncoded term.

Summary of Adverse Events—Phase 2/3 First 6610 Participants—Phase 2/3

Table 14 presents a summary of the first 6610 participants reporting atleast 1 AE from Dose 1 to 1 month after Dose 2.

The number of participants who reported at least 1 AE was similar in theBNT162b2 group as compared with the placebo group. Severe AEs, SAEs, andAEs leading to withdrawal were reported by 1.1%, 0.5%, and 0.2%,respectively, in both groups. In the younger and older age groups, thenumbers of participants who reported at least 1 AE from Dose 1 to 1month after Dose 2 were similar in the BNT162b2 group and thecorresponding placebo group. Rates of related AEs, severe AEs, SAEs, andAEs leading to withdrawal in the younger and older age groups were alsosimilar to the corresponding placebo group.

The first 6610 participants who reported at least 1 AE from Dose 1 tothe data cutoff date in the BNT162b2 group and the placebo group weresimilar to those in the corresponding groups at 1 month after Dose 2(Table 14). From 1 month after Dose 2 to the data cutoff date, 4additional participants in the younger age group (3 in BNT162b2 and 1 inplacebo) and 10 additional participants in the older age group (3 inBNT162b2 and 7 in placebo) reported at least 1 AE. There were noadditional related AEs, severe AEs, SAEs, or AEs leading to withdrawalreported in either group.

TABLE 14 Number (%) of Subjects Reporting at Least 1 Adverse Event FromDose 1 to 1 Month After Dose 2 - ~6000 Subjects for Phase 2/3 Analysis -Safety Population Vaccine Group (as Administered) BNT162b2 (30 μg)Placebo (N^(a) = 3314) (N^(a) = 3296) Adverse Event n^(b) (%) n^(b) (%)Any event 374 (11.3) 316 (9.6) Related^(c) 135 (4.1) 68 (2.1) Severe 35(1.1) 19 (0.6) Life-threatening 4 (0.1) 7 (0.2) Any serious adverseevent 18 (0.5) 17 (0.5) Related^(c) 0 0 Severe 9 (0.3) 8 (0.2)Life-threatening 4 (0.1) 7 (0.2) Any adverse event leading 6 (0.2) 5(0.2) to withdrawal Related^(c) 2 (0.1) 1 (0.0) Severe 2 (0.1) 1 (0.0)Life-threatening 1 (0.0) 2 (0.1) Death 0 0 ^(a)N = number of subjects inthe specified group. This value is the denominator for the percentagecalculations. ^(b)n = Number of subjects reporting at least 1 occurrenceof the specified adverse event category. For “any event”, n = the numberof subjects reporting at least 1 occurrence of any adverse event.^(c)Assessed by the investigator as related to investigational product.

All Participants—Phase 2/3

From Dose 1 to the data cutoff date, the number of overall participantswho reported at least 1 AE was higher in the BNT162b2 group as comparedwith the placebo group. Severe AEs, SAEs, and AEs leading to withdrawalwere reported by ≤0.8%, 0.3%, and 0.1%, respectively, in both groups.Discontinuations due to related AEs were reported in 6 participants inthe BNT162b2 group and 4 participants in the placebo group.

Three Phase 3 participants died: 1 participant in the BNT162b2 group and2 participants in the placebo group. The participant in the BNT162b2group who died experienced an SAE of arteriosclerosis which was assessedby the investigator as not related to study intervention.

In the younger age group, the number of participants who reported atleast 1 AE was 1920 (18.1%) and 880 (8.3%) in the BNT162b2 and placebogroups, respectively. In the older age group, the number of participantswho reported at least 1 AE was 1166 (14.9%) and 582 (7.4%) in theBNT162b2 and placebo groups, respectively.

Analysis of Adverse Events—Phase 2/3 Adverse Events by System OrganClass and Preferred Term—Phase 2/3 First 6610 Participants—Phase 2/3

There are no Tier 1 AEs identified for this program.

There were no Tier 2 AEs (defined as an event rate ≥1.0% in any vaccinegroup [PT level]) reported from Dose 1 to 1 month after Dose 2.

Most AEs reported up to 1 month after Dose 2 overall were reactogenicityand in the SOCs of general disorders and administration site conditions(81 [2.4%] in the BNT162b2 group and 57 [1.7%] in the placebo group),musculoskeletal and connective tissue disorders (81 [2.4%] in theBNT162b2 group and 56 [1.7%] in the placebo group), infections andinfestations (56 [1.7%] in the BNT162b2 group and 48 [1.5%] in theplacebo group), and gastrointestinal disorders (54 [1.6%] in theBNT162b2 group and 41 [1.2%] in the placebo group) (Table 15). In theyounger BNT162b2 group, rates of AEs in these SOCs were: generaldisorders and administration site conditions (54 [3.0%]),musculoskeletal and connective tissue disorders (53 [3.0%]), infectionsand infestations (31 [1.7%]), and gastrointestinal disorders (32[1.8%]). In the older BNT162b2 group, rates of AEs in these SOCs were:general disorders and administration site conditions (27 [1.8%]),musculoskeletal and connective tissue disorders (28 [1.8%]), infectionsand infestations (25 [1.6%]), and gastrointestinal disorders (22[1.4%]).

In the BNT162b2 group, the most frequently reported AEs by PT overallwere injection site pain (30 [0.9%]), headache (30 [0.9%]), and fatigue(27 [0.8%]) (Table 15), and during this time period (from Dose 1 to 1month after Dose 2) most of these AEs were reported during the e-diary 1week reporting period. The majority of these PTs were reported in theyounger age group: headache (21[1.2%]), and fatigue (17 [1.0%]).Injection site pain was reported at a similar frequency in the younger(16 [0.9%]) and older (14 [0.9%]) age groups.

In the BNT162b2 group, there were 10 (0.3%) participants who reported anAE of lymphadenopathy: 6 in the younger age group and 4 in the older agegroup compared to none in the placebo group; 1 (0.1%) was male and 9(0.5%) were females. AEs of lymphadenopathy occurred in the arm and neckregion (in axillary, left axillary, left para clavicular, left supraclavicular, bilateral cervical, or unspecified lymph nodes). Mostlymphadenopathy events were reported within 2 to 4 days aftervaccination (2 events were reported 8 days after vaccination). Five ofthe events lasted 54 days, 3 events lasted between 12 to 16 days, and 2events were ongoing at the time of the data cutoff date.

In the younger age group, an AE of angioedema 13 days after Dose 1 (botheyes) and hypersensitivity (allergy attack [no additional informationavailable at the time of this report], unrelated to study intervention)were reported in 1 participant each (BNT162b2 group), and an AE of drughypersensitivity (oral penicillin reaction) was reported in 1participant (placebo). None of these events were assessed by theinvestigator as related to study intervention. Three participants in theyounger BNT162b2 group reported appendicitis compared to 1 participantin the older placebo group with perforated appendicitis; all wereassessed by the investigator as unrelated to study intervention.

TABLE 15 Number (%) of Subjects Reporting at Least 1 Adverse Event FromDose 1 to 1 Month After Dose 2, by System Organ Class and PreferredTerm - ~6000 Subjects for Phase 2/3 Analysis - Safety Population VaccineGroup (as Administered) BNT162b2 (30 μg) Placebo System Organ Class(N^(a) = 3314) (N^(a) = 3296) Preferred Term n^(b) (%) (95% CI^(c))n^(b) (%) (95% CI^(c)) Any event 374 (11.3) (10.2, 12.4) 316 (9.6) (8.6, 10.6) BLOOD AND LYMPHATIC SYSTEM 14 (0.4) (0.2, 0.7) 0 (0.0, 0.1)DISORDERS Lymphadenopathy 10 (0.3) (0.1, 0.6) 0 (0.0, 0.1) Anaemia 2(0.1) (0.0, 0.2) 0 (0.0, 0.1) Iron deficiency anaemia 1 (0.0) (0.0, 0.2)0 (0.0, 0.1) Lymph node pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) CARDIACDISORDERS 6 (0.2) (0.1, 0.4) 4 (0.1) (0.0, 0.3) Atrial fibrillation 1(0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Mitral valve incompetence 0 (0.0,0.1) 2 (0.1) (0.0, 0.2) Palpitations 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0,0.2) Acute coronary syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Acutemyocardial infarction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Angina pectoris 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Angina unstable 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Atrial flutter 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cardiac failurecongestive 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery disease 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery dissection 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Coronary artery occlusion 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Left atrial enlargement 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Leftventricular hypertrophy 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Mitral valveprolapse 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tachycardia 1 (0.0) (0.0, 0.2)0 (0.0, 0.1) CONGENITAL, FAMILIAL AND 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)GENETIC DISORDERS Congenital cystic kidney 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) disease EAR AND LABYRINTH 7 (0.2) (0.1, 0.4) 6 (0.2) (0.1, 0.4)DISORDERS Vertigo 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Vertigopositional 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Ear discomfort 2 (0.1)(0.0, 0.2) 0 (0.0, 0.1) Deafness unilateral 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) Ear pain 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tinnitus 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Tympanic membrane 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)perforation ENDOCRINE DISORDERS 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Goitre 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypogonadism 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) EYE DISORDERS 8 (0.2) (0.1, 0.5) 6 (0.2) (0.1, 0.4) Cataract 1(0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Chalazion 2 (0.1) (0.0, 0.2) 0 (0.0,0.1) Vision blurred 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Blepharitis 0 (0.0,0.1) 1 (0.0) (0.0, 0.2) Conjunctival hyperaemia 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Dacryostenosis acquired 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Diplopia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Eye pain 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) Lacrimation increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Retinaldetachment 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vitreous detachment 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) GASTROINTESTINAL 54 (1.6) (1.2, 2.1) 41 (1.2)(0.9, 1.7) DISORDERS Diarrhoea 17 (0.5) (0.3, 0.8) 15 (0.5) (0.3, 0.7)Nausea 12 (0.4) (0.2, 0.6) 5 (0.2) (0.0, 0.4) Toothache 5 (0.2) (0.0,0.4) 2 (0.1) (0.0, 0.2) Vomiting 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3)Abdominal pain 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Constipation 2(0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dyspepsia 1 (0.0) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Gastrooesophageal reflux 0 (0.0, 0.1) 3 (0.1) (0.0, 0.3)disease Odynophagia 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Dental caries1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Inguinal hernia 1 (0.0) (0.0, 0.2)1 (0.0) (0.0, 0.2) Small intestinal 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2)obstruction Abdominal adhesions 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Abdominal pain upper 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Abdominal rigidity1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Angular cheilitis 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) Colitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diverticularperforation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Faeces soft 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Gastric ulcer haemorrhage 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) Gastrointestinal disorder 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gingivaldiscomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Haematochezia 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Hiatus hernia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Hypoaesthesia oral 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Intestinalobstruction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Irritable bowel syndrome 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Mouth ulceration 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Parotid duct obstruction 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Rectal haemorrhage 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Salivary glandcalculus 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tongue discomfort 0 (0.0, 0.1)1 (0.0) (0.0, 0.2) Tongue ulceration 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)GENERAL DISORDERS AND 81 (2.4) (1.9, 3.0) 57 (1.7) (1.3, 2.2)ADMINISTRATION SITE CONDITIONS Fatigue 27 (0.8) (0.5, 1.2) 17 (0.5)(0.3, 0.8) Injection site pain 30 (0.9) (0.6, 1.3) 14 (0.4) (0.2, 0.7)Chills 15 (0.5) (0.3, 0.7) 7 (0.2) (0.1, 0.4) Injection site erythema 10(0.3) (0.1, 0.6) 6 (0.2) (0.1, 0.4) Pyrexia 13 (0.4) (0.2, 0.7) 2 (0.1)(0.0, 0.2) Injection site swelling 4 (0.1) (0.0, 0.3) 4 (0.1) (0.0, 0.3)Pain 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Malaise 3 (0.1) (0.0, 0.3) 1(0.0) (0.0, 0.2) Injection site bruising 1 (0.0) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Injection site reaction 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1)Asthenia 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Injection site pruritus 1(0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Chest discomfort 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Chest pain 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Face oedema1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Inflammation 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) Injection site discolouration 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Injection site discomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Injection sitehyperaesthesia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Non-cardiac chest pain 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Swelling 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Unevaluable event 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaccination sitenodule 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vascular stent occlusion 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) HEPATOBILIARY DISORDERS 2 (0.1) (0.0, 0.2) 1(0.0) (0.0, 0.2) Cholelithiasis 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1)Cholecystitis acute 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) IMMUNE SYSTEMDISORDERS 4 (0.1) (0.0, 0.3) 6 (0.2) (0.1, 0.4) Seasonal allergy 1 (0.0)(0.0, 0.2) 3 (0.1) (0.0, 0.3) Food allergy 1 (0.0) (0.0, 0.2) 1 (0.0)(0.0, 0.2) Allergy to vaccine 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Anaphylactic reaction 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Drughypersensitivity 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hypersensitivity 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Milk allergy 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) INFECTIONS AND 56 (1.7) (1.3, 2.2) 48 (1.5) (1.1, 1.9) INFESTATIONSUrinary tract infection 10 (0.3) (0.1, 0.6) 7 (0.2) (0.1, 0.4) Sinusitis8 (0.2) (0.1, 0.5) 1 (0.0) (0.0, 0.2) Diverticulitis 3 (0.1) (0.0, 0.3)4 (0.1) (0.0, 0.3) Tooth infection 2 (0.1) (0.0, 0.2) 3 (0.1) (0.0, 0.3)Otitis media 0 (0.0, 0.1) 4 (0.1) (0.0, 0.3) Upper respiratory tractinfection 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Appendicitis 3 (0.1)(0.0, 0.3) 0 (0.0, 0.1) Cellulitis 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2)Ear infection 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Herpes zoster 2(0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Pneumonia 1 (0.0) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Tonsillitis 0 (0.0, 0.1) 3 (0.1) (0.0, 0.3) Conjunctivitis 1(0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Gastroenteritis 0 (0.0, 0.1) 2 (0.1)(0.0, 0.2) Infected bite 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Otitis externa1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Otitis media acute 1 (0.0) (0.0,0.2) 1 (0.0) (0.0, 0.2) Skin infection 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0,0.2) Tooth abscess 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Acute sinusitis 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Appendicitis perforated 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Cystitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Dermatitisinfected 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Eye infection 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Folliculitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fungalinfection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fungal skin infection 0 (0.0,0.1) 1 (0.0) (0.0, 0.2) Genital herpes 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Genital herpes simplex 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gingivitis 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Hordeolum 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Kidney infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lower respiratory tractinfection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Oral candidiasis 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Oral fungal infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Parotitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Peritoneal abscess 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Peritonitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Pharyngitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Pharyngitis streptococcal 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Respiratory tract infection viral 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Rhinitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sepsis1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Skin bacterial infection 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Soft tissue infection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Subcutaneous abscess 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vulvovaginalcandidiasis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vulvovaginal mycoticinfection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INJURY, POISONING AND 28 (0.8)(0.6, 1.2) 42 (1.3) (0.9, 1.7) PROCEDURAL COMPLICATIONS Fall 8 (0.2)(0.1, 0.5) 16 (0.5) (0.3, 0.8) Arthropod bite 5 (0.2) (0.0, 0.4) 3 (0.1)(0.0, 0.3) Muscle strain 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Contusion2 (0.1) (0.0, 0.2) 4 (0.1) (0.0, 0.3) Skin abrasion 1 (0.0) (0.0, 0.2) 4(0.1) (0.0, 0.3) Skin laceration 0 (0.0, 0.1) 4 (0.1) (0.0, 0.3) Anklefracture 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Joint dislocation 2 (0.1)(0.0, 0.2) 1 (0.0) (0.0, 0.2) Tooth fracture 1 (0.0) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Fibula fracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Footfracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Joint injury 1 (0.0)(0.0, 0.2) 1 (0.0) (0.0, 0.2) Ligament sprain 1 (0.0) (0.0, 0.2) 1 (0.0)(0.0, 0.2) Limb injury 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Meniscus injury 2(0.1) (0.0, 0.2) 0 (0.0, 0.1) Muscle rupture 0 (0.0, 0.1) 2 (0.1) (0.0,0.2) Rib fracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Bone contusion 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Chest injury 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Corneal abrasion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Craniocerebralinjury 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Forearm fracture 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Hand fracture 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Humerusfracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lumbar vertebral fracture 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Muscle injury 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Postoperative ileus 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Procedural pain1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Radius fracture 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Road traffic accident 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sunburn1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tendon rupture 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Thermal burn 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) INVESTIGATIONS 9(0.3) (0.1, 0.5) 4 (0.1) (0.0, 0.3) Body temperature increased 2 (0.1)(0.0, 0.2) 1 (0.0) (0.0, 0.2) Blood glucose increased 2 (0.1) (0.0, 0.2)0 (0.0, 0.1) Blood cholesterol increased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Blood pressure increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Bloodtriglycerides increased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Cardiac stresstest abnormal 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Electrocardiogram QTprolonged 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Heart rate increased 0 (0.0,0.1) 1 (0.0) (0.0, 0.2) Weight decreased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)White blood cell count increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)METABOLISM AND NUTRITION 12 (0.4) (0.2, 0.6) 7 (0.2) (0.1, 0.4)DISORDERS Decreased appetite 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1)Hypercholesterolaemia 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Type 2diabetes mellitus 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dehydration 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Diabetes mellitus inadequate control 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Dyslipidaemia 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) Glucose tolerance impaired 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Hyperlipidaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypertriglyceridaemia 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypoglycaemia 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Hypokalaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Iron deficiency 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Obesity 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Vitamin D deficiency 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) MUSCULOSKELETAL AND81 (2.4) (1.9, 3.0) 56 (1.7) (1.3, 2.2) CONNECTIVE TISSUE DISORDERSArthralgia 20 (0.6) (0.4, 0.9) 15 (0.5) (0.3, 0.7) Myalgia 20 (0.6)(0.4, 0.9) 12 (0.4) (0.2, 0.6) Back pain 8 (0.2) (0.1, 0.5) 8 (0.2)(0.1, 0.5) Pain in extremity 10 (0.3) (0.1, 0.6) 4 (0.1) (0.0, 0.3) Neckpain 5 (0.2) (0.0, 0.4) 5 (0.2) (0.0, 0.4) Muscle spasms 4 (0.1) (0.0,0.3) 2 (0.1) (0.0, 0.2) Musculoskeletal pain 2 (0.1) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Osteoarthritis 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2)Tendonitis 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Arthritis 1 (0.0) (0.0,0.2) 1 (0.0) (0.0, 0.2) Flank pain 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2)Joint effusion 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Plantar fasciitis 0 (0.0,0.1) 2 (0.1) (0.0, 0.2) Bursitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Exostosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Limb discomfort 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Muscle twitching 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Muscular weakness 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Musculoskeletaldiscomfort 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Musculoskeletal stiffness 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Osteitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Osteopenia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Pain in jaw 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Rotator cuff syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Tenosynovitis stenosans 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Torticollis 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMS BENIGN, MALIGNANT 4 (0.1) (0.0,0.3) 3 (0.1) (0.0, 0.3) AND UNSPECIFIED (INCL CYSTS AND POLYPS) Basalcell carcinoma 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Adenocarcinomagastric 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Breast cancer 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Seborrhoeic keratosis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Uterine leiomyoma 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaginal neoplasm 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEM DISORDERS 44 (1.3) (1.0,1.8) 32 (1.0) (0.7, 1.4) Headache 30 (0.9) (0.6, 1.3) 24 (0.7) (0.5,1.1) Dizziness 3 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) Migraine 3 (0.1)(0.0, 0.3) 1 (0.0) (0.0, 0.2) Paraesthesia 3 (0.1) (0.0, 0.3) 1 (0.0)(0.0, 0.2) Syncope 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Burningsensation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cervical radiculopathy 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Dysgeusia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Hypoaesthesia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Radiculopathy 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Sciatica 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Transient ischaemic attack 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tremor 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) PSYCHIATRIC DISORDERS 13 (0.4) (0.2, 0.7)13 (0.4) (0.2, 0.7) Anxiety 4 (0.1) (0.0, 0.3) 4 (0.1) (0.0, 0.3)Depression 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Anxiety disorder 0(0.0, 0.1) 2 (0.1) (0.0, 0.2) Irritability 2 (0.1) (0.0, 0.2) 0 (0.0,0.1) Mental status changes 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Bipolardisorder 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Depressed mood 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Disorientation 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Gastrointestinal somatic 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) symptomdisorder Insomnia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Mental disorder 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Mood swings 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Panic reaction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sleep disorder 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Suicidal ideation 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) RENAL AND URINARY 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3)DISORDERS Acute kidney injury 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Chronickidney disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Dysuria 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Haematuria 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Nephrolithiasis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Urinary retention 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) REPRODUCTIVE SYSTEM AND 4 (0.1) (0.0, 0.3)2 (0.1) (0.0, 0.2) BREAST DISORDERS Dysmenorrhoea 3 (0.1) (0.0, 0.3) 0(0.0, 0.1) Cervical dysplasia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Menorrhagia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Prostatitis 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) RESPIRATORY, THORACIC AND 21 (0.6) (0.4, 1.0) 26 (0.8)(0.5, 1.2) MEDIASTINAL DISORDERS Oropharyngeal pain 4 (0.1) (0.0, 0.3) 7(0.2) (0.1, 0.4) Cough 5 (0.2) (0.0, 0.4) 5 (0.2) (0.0, 0.4) Rhinitisallergic 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Rhinorrhoea 1 (0.0) (0.0,0.2) 4 (0.1) (0.0, 0.3) Nasal congestion 2 (0.1) (0.0, 0.2) 1 (0.0)(0.0, 0.2) Dyspnoea 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dyspnoeaexertional 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Upper-airway coughsyndrome 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Acute respiratory failure 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Allergic respiratory disease 0 (0.0, 0.1)1 (0.0) (0.0, 0.2) Asthma 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Bronchospasm 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Chronic obstructive pulmonary 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) disease Productive cough 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Pulmonary embolism 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sinuscongestion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Throat irritation 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) SKIN AND SUBCUTANEOUS 32 (1.0) (0.7, 1.4) 19(0.6) (0.3, 0.9) TISSUE DISORDERS Dermatitis contact 10 (0.3) (0.1, 0.6)3 (0.1) (0.0, 0.3) Rash 7 (0.2) (0.1, 0.4) 2 (0.1) (0.0, 0.2) Erythema 3(0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Urticaria 2 (0.1) (0.0, 0.2) 2 (0.1)(0.0, 0.2) Dermatitis 1 (0.0) (0.0. 0.2) 2 (0.1) (0.0, 0.2) Pruritus 3(0.1) (0.0. 0.3) 0 (0.0, 0.1) Hyperhidrosis 0 (0.0, 0.1) 2 (0.1) (0.0.0.2) Macule 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0. 0.2) Angioedema 1 (0.0)(0.0, 0.2) 0 (0.0. 0.1) Dermatitis atopic 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Dermatitis bullous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Drug eruption 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Ecchymosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Hangnail 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Papule 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) Rash erythematous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Rashmaculo-papular 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Rosacea 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Skin discolouration 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Skin ulcer 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) SURGICAL AND MEDICAL 5 (0.2)(0.0, 0.4) 4 (0.1) (0.0, 0.3) PROCEDURES Dental care 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) Dental operation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Gingival operation 1 (0.0) (0.0. 0.2) 0 (0.0, 0.1) Hip surgery 1 (0.0)(0.0. 0.2) 0 (0.0, 0.1) Inguinal hernia repair 1 (0.0) (0.0. 0.2) 0(0.0, 0.1) Laryngeal operation 0 (0.0. 0.1) 1 (0.0) (0.0, 0.2)Postoperative care 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sclerotherapy 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Tooth extraction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)VASCULAR DISORDERS 8 (0.2) (0.1, 0.5) 15 (0.5) (0.3, 0.7) Hypertension 5(0.2) (0.0, 0.4) 6 (0.2) (0.1, 0.4) Haematoma 1 (0.0) (0.0, 0.2) 3 (0.1)(0.0, 0.3) Aortic aneurysm 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Deep veinthrombosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Essential hypertension 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypotension 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) LYMPHOEDEMA 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lymphorrhoea 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Orthostatic hypotension 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Raynaud's phenomenon 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Note:MedDRA (v23.0) coding dictionary applied. ^(a)N = number of subjects inthe specified group. This value is the denominator for the percentagecalculations. ^(b)n = Number of subjects reporting at least 1 occurrenceof the specified event. For “any event”, n = number of subjectsreporting at least 1 occurrence of any event. ^(c)Exact 2-sided CI basedon the Clopper and Pearson method.

All Participants—Phase 2/3

For all 36,855 participants up to the data cutoff date, there were atotal of 121 (0.7%) participants in the BNT162b2 group and 51 (0.3%)participants in the placebo group with at least 1 uncoded term. As aresult, uncoded terms are also present in other AE tables summarized bySOC and PT.

From Dose 1 to the data cutoff date, the number of overall participantswho reported at least 1 AE was higher in the BNT162b2 group (3086[16.8%]) as compared with the placebo group (1462 [7.9%]). Most AEsreported in all participants from Dose 1 to the data cutoff date werereactogenicity and in the SOCs of general disorders and administrationsite conditions (1941 [10.5%] in the BNT162b2 group and 438 [2.4%] inthe placebo group), musculoskeletal and connective tissue disorders (742[4.0%] in the BNT162b2 group and 227 [1.2%] in the placebo group), andnervous system disorders (567 [3.1%] in the BNT162b2 group and 251[1.4%] in the placebo group). In the BNT162b2 group, the most frequentlyreported AEs by PT were injection site pain (1222 [6.6%]), pyrexia (504[2.7%]), fatigue (481 [2.6%]), headache (470 [2.6%]), chills (458[2.5%]), and myalgia (454 [2.5%]). The majority of these PTs werereported in the younger age group: injection site pain (787 [7.4%]),pyrexia (351 [3.3%]), fatigue (309 [2.9%]), headache (303 [2.9%]),chills (316 [3.0%]), and myalgia (304 [2.9%]). Beyond the first 6610participants, events related to reactogenicity are no longer reportedusing an e-diary but are instead reported as AEs. Therefore, a post hocanalysis was conducted to evaluate if the imbalance in AEs observed inthe overall participants from Dose 1 to the data cutoff date but notobserved in the first 6610 participants from Dose 1 to 1 month afterDose 2 was attributed to reactogenicity events. The analysis examinedthe AEs reported within 7 days after each dose, which represented thereactogenicity reporting period. The time period was chosen because manyAEs were reported in the SOCs of general disorders and administrationsite conditions, musculoskeletal and connective tissue disorders, andnervous system disorders, which contains AEs consistent withreactogenicity events, and could only be attributed to reactogenicity ifthey occurred during this time period as opposed to occurring up to amonth from each dose.

From Dose 1 to 7 days after Dose 1 (as of the data cutoff date), 1494(8.1%) participants reported at least 1 AE in the BNT162b2 group, whichrepresented approximately half of the total number of the 3086 [16.8%]participants who reported at least 1 AE up to the data cutoff date. Inthe placebo group, 555 (3.0%) participants reported at least 1 AE fromDose 1 to 7 days after Dose 1, compared with the total number of 1462(7.9%) participants who reported at least 1 AE up to the data cutoffdate.

From Dose 2 to 7 days after Dose 2 (as of the data cutoff date), 1165(6.3%) participants reported at least 1 AE in the BNT162b2 group, whichrepresented approximately 38% of the total number of the 3086 [16.8%]participants who reported at least 1 AE up to the data cutoff date. FromDose 2 to 7 days after Dose 2, fewer participants reported AEs in theplacebo group than the BNT162b2 group. In the placebo group, 268 (1.5%)participants reported at least 1 AE from Dose 2 to 7 days after Dose 2,compared with the total number of 1462 (7.9%) participants who reportedat least 1 AE up to the data cutoff date.

AEs were reported from Dose 1 to 7 days after Dose 1 in the SOC ofgeneral disorders and administration site conditions (1127 [6.1%] in theBNT162b2 group and 251 [1.4%] in the placebo group), which representedmore than half of the total number of participants reporting at least 1AE in this SOC (1941 [10.5%] in the BNT162b2 group and 438 [2.4%] in theplacebo group) up to the data cutoff date. Musculoskeletal andconnective tissue disorders (252 [1.4%] in the BNT162b2 group and 76[0.4%] in the placebo group) and nervous system disorders (220 [1.2%] inthe BNT162b2 group and 115 [0.6%] in the placebo group) were alsocommonly reported, representing a smaller proportion of the total numberof participants reporting AEs for these SOCs. In the BNT162b2 group, themost frequently reported AEs from Dose 1 to 7 days after Dose 1 by PTwere injection site pain (881 [4.8%]), fatigue (231 [1.3%]), headache(181 [1.0%]), myalgia (147 [0.8%]), pyrexia (110 [0.6%]), and chills(100 [0.5%]). The majority of these PTs were reported in the younger agegroup: injection site pain (566 [5.3%]), fatigue (153 [1.4%]), headache(118 [1.1%]), myalgia (99 [0.9%]), pyrexia (82 [0.8%]), and chills (75[0.7%]). Injection site pain reported from Dose 1 to 7 days after Dose 1(881 [4.8%]) represented a large proportion of the total participantswho reported AEs for this PT (1222 [6.6%]).

AEs were reported from Dose 2 to 7 days after Dose 2 in the SOCs ofgeneral disorders and administration site conditions (828 [4.5%] in theBNT162b2 group and 93 [0.5%] in the placebo group), musculoskeletal andconnective tissue disorders (377 [2.0%] in the BNT162b2 group and 38[0.2%] in the placebo group), and nervous system disorders (294 [1.6%]in the BNT162b2 group and 40 [0.2%] in the placebo group).Musculoskeletal and connective tissue disorders and nervous systemdisorders reported from Dose 2 to 7 days after Dose 2 represented atleast half of the total number of participants who reported at least 1AE in these SOCs.

In the BNT162b2 group, the most frequently reported AEs from Dose 2 to 7days after Dose 2 by PT were pyrexia (375 [2.0%]), chills (327 [1.8%]),injection site pain (313 [1.7%]), myalgia (282 [1.5%]), headache (258[1.4%]), and fatigue (227 [1.2%]). The majority of these PTs werereported in the younger age group: pyrexia (251 [2.4%]), chills (216[2.0%]), myalgia (185 [1.7%]), injection site pain (183 [1.7%]),headache (154 [1.5%]), and fatigue (134 [1.3%]). AEs for most of thesePTs reported from Dose 2 to 7 days after Dose 2 represented at leasthalf of the total number of participants who reported an AE for thesePTs: pyrexia (504 [2.7%]), chills (458 [2.5%]), myalgia (454 [2.5%]),headache (470 [2.6%]), and fatigue (481 [2.6%]).

Overall, AEs reported from Dose 1 to 7 days after Dose 1 and from Dose 2to 7 days after Dose 2 were largely attributable to reactogenicityevents. This observation provides a reasonable explanation for thegreater rates of AEs observed overall in the BNT162b2 group comparedwith the placebo group.

From Dose 1 to the data cutoff date, there were a total of 44 (0.2%)participants in the BNT162b2 group who reported an AE oflymphadenopathy, inclusive of those reported in the first 6610participants (10 [0.3%]). Up to the data cutoff date, 34 additionalparticipants in the BNT162b2 group and 4 additional participants in theplacebo group reported an AE of lymphadenopathy. In the BNT162b2 group,lymphadenopathy was reported in 34 (0.3%) participants in the youngerage group and 10 (0.1%) participants in the older age group compared to4 (0.0%) in the placebo group (3 in the younger age group and 1 in theolder age group). Lymphadenopathy occurred predominantly in the arm andneck region with most events reported in left axillary lymph node(s).Most lymphadenopathy events occurred after Dose 2, ≤3 days after Dose 1or Dose 2, were Grade 1 or Grade 2 in severity, and 32 of 48 events wereresolved by the data cutoff date. In 1 participant in the youngerBNT162b2 age group, Grade 1 lymphadenopathy (swollen right axillarylymph nodes) was an immediate AE, which occurred after Dose 1 and wascontinuing at the data cutoff date.

In the younger age group, there was 1 participant each with an AE ofsuspected COVID-19 in the BNT162b2 (SAE) and placebo groups.

In the BNT162b2 group, 6 participants reported immunization reactions(vaccine reaction or systemic vaccine reaction [no additionalinformation currently available at the time of this report]) assessed asrelated to study intervention. Three participants reported drughypersensitivity in the BNT162b2 group in addition to the drughypersensitivity in a participant in the placebo group. Drughypersensitivity (allergic reaction) was assessed as related in 1participant in the BNT162b2 group and drug hypersensitivity (drugallergy or allergic reaction to dipyrone) was assessed as unrelated tostudy intervention in 2 participants in the BNT162b2 group.

Nineteen (0.1%) participants in the BNT162b2 group (14 in the youngerage group and 5 in the older age group reported at least 1 vaccinecomplication (most were descriptive of reactogenicity events) comparedto none in the placebo group. All were assessed as related to the studyintervention and included: post vaccination myalgia, fever, body aches,headache, chills, nausea, adverse reaction, arthralgia, fatigue, aches,muscle aches, malaise, and sore left shoulder. Most events were Grade 1,started within 3 days of vaccination, and lasted from 1 to 3 days.

In addition to the 4 participants with appendicitis (including 1appendicitis perforated in the placebo group) in the first 6610participants, there were an additional 3 participants with appendicitis(including 1 participant with appendicitis perforated) reported in theBNT162b2 group from Dose 1 through the data cutoff date for allparticipants. Therefore, a total of 6 participants in the BNT162b2 groupreported appendicitis (including 1 appendicitis perforated) with 4 inthe younger age group and 2 in the older age group, and 1 participant inthe placebo group (older age group) reported appendicitis (perforated).All events were severe or life-threatening and none were assessed asrelated to study intervention.

Related Adverse Events by System Organ Class and Preferred Term—Phase2/3 First 6610 Participants—Phase 2/3

From Dose 1 to 1 month after Dose 2, 135 (4.1%) participants reported atleast 1 AE assessed as related by the investigator in the BNT162b2group, and 68 (2.1%) participants reported at least 1 related AE in theplacebo group. Most related AEs were reactogenicity events and in theSOC of general disorders and administration site conditions (69 [2.1%]in the BNT162b2 group and 40 [1.2%] in the placebo group). The AEs oflymphadenopathy reported in 8 of 10 participants were assessed by theinvestigator as related to study intervention.

All Participants—Phase 2/3

From Dose 1 to the data cutoff date, 2303 (12.5%) participants in theBNT162b2 group and 593 (3.2%) participants in the placebo group reportedat least 1 AE assessed as related by the investigator, inclusive of therelated AEs for the first 6610 participants. Most related AEs werereactogenicity events and in the SOC of general disorders andadministration site conditions (1869 [10.1%] in the BNT162b2 group and365 [2.0%] in the placebo group).

The AEs of lymphadenopathy reported in 30 of 44 participants in theBNT162b2 group and 2 of 4 participants in the placebo group wereassessed by the investigator as related to study intervention.

In the BNT162b2 group, based on all information currently available atthe time of this report:

Six participants reported immunization reaction (vaccine reaction orsystemic vaccine reaction) assessed as related to the studyintervention. In most participants, immunization reactions occurred 1 or2 days after Dose 2, lasted 2 or 3 days (1 participant was recovering atdata cutoff date), and were Grade 1 or Grade 2 in severity. In 1participant, immunization reactions (systemic vaccine reactions)occurred 2 days after Dose 1 (Grade 1) and lasted 2 days, and 1 dayafter Dose 2 (Grade 3) and lasted 4 days.

One participant reported an AE each of drug hypersensitivity (allergicreaction), urticaria (allergic reaction), and headache, which were allGrade 2 and assessed by the investigator as related to studyintervention. The AEs of drug hypersensitivity and urticaria bothoccurred within 1 day after Dose 1 and resolved that same day. The AE ofheadache occurred the following day after vaccination and lasted 4 days.

Immediate Adverse Events—Phase 2/3 First 6610 Participants—Phase 2/3

After Dose 1, ≤0.3% of participants reported immediate AEs. Mostimmediate AEs were in the SOC of general disorders and administrationsite conditions and were events related to injection site reactions(injection site pain, injection site erythema and injection siteswelling).

After Dose 2, 0.1% of participants in each group reported immediate AEs.Most immediate AEs were in the SOC of general disorders andadministration site conditions and were events related to injection sitereactions (injection site pain, injection site hyperaesthesia, andinjection site pruritus).

After either dose of BNT162b2, no participant reported an immediateallergic reaction to the vaccine.

All Participants—Phase 2/3

After Dose 1, 0.3% of participants in each group reported immediate AEs.Most immediate AEs were in the SOC of general disorders andadministration site conditions and most events were related to injectionsite reactions with injection site pain most frequently reported (40[0.2%] participants in the BNT162b2 group and 27 (0.1%) participants inthe placebo group). One participant had an immediate AE oflymphadenopathy after Dose 1. All other immediate AEs were reported by53 participants each in the BNT162b2 group.

After Dose 2, 0.1% of participants in each group reported immediate AEs.Most immediate AEs were in the SOC of general disorders andadministration site conditions and most events were injection sitereactions with injection site pain most frequently reported (10 [0.1%]participants in the BNT162b2 group and 7 [0.0%] participants in theplacebo group). All other immediate AEs were reported by 52 participantseach. After either dose of BNT162b2, no participant reported animmediate allergic reaction to the vaccine.

Severe or Life-Threatening Adverse Events—Phase 2/3 First 6610Participants—Phase 2/3

From Dose 1 to 1 month after Dose 2, severe AEs reported were reportedby 35 (1.1%) participants in the BNT162b2 group and 19 (0.6%) in theplacebo group.

Four (0.1%) participants in the BNT162b2 group and 7 (0.2%) participantsin the placebo group had at least 1 life-threatening AE from Dose 1 to 1month after Dose 2. None of these events were assessed by theinvestigator as related to study intervention.

In the BNT162b2 group:

One participant from Phase 2 had a severe event of gastricadenocarcinoma (SAE), which is discussed in a previous section.

Two participants had severe events of appendicitis: 1 event began 9 daysafter Dose 1 and the other event began 15 days after Dose 2 (SAEs) whichwere assessed by the investigator as not related to study intervention.

One participant had 2 life-threatening AEs of appendicitis andperitoneal abscess 7 days after Dose 1 (both SAEs); both events wereassessed by the investigator as not related to study intervention.

One participant had 8 severe events: anemia, cardiac failure congestive,abdominal adhesions, sepsis, hypokalaemia, mental status changes, acutekidney injury, and acute respiratory failure (all SAEs). None of theevents were assessed by the investigator as related to studyintervention.

All Participants—Phase 2/3

Severe AEs reported up to the data cutoff date, inclusive of thosediscussed for the first 6610 participants, were reported by 142 [0.8%]participants in the BNT162b2 group and 70 (0.4%) in the placebo group.Additional events included:

Two participants in the BNT162b2 group had a severe event each ofappendicitis: 1 event began 17 days after Dose 1 and the other eventbegan 11 days after Dose 1 (SAE) which were assessed by the investigatoras not related to study intervention. One participant in the BNT162b2group had a severe event of perforated appendicitis on the same dayafter Dose 1 (SAE) which was assessed by the investigator as not relatedto study intervention.

Nine participants (0.0%) in the BNT162b2 group and 12 (0.1%)participants in the placebo group had at least 1 life-threatening AEfrom Dose 1 to the data cutoff date, inclusive of those discussed forthe first 6610 participants. None of these events were assessed by theinvestigator as related to study intervention.

Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals,and Other Significant Adverse Events—Phase 2/3 Deaths—Phase 2/3

There were 3 Phase 3 participants (1 in the BNT162b2 group and 2 in theplacebo group) who died through the data cutoff date of 6 Oct. 2020.None of these deaths were among the first 6610 participants (Table 14)and none were assessed by the investigator as related to studyintervention.

One participant in the older BNT162b2 group experienced a Grade 4 SAE ofarteriosclerosis 4 days after Dose 1 and died 15 days after Dose 1.

One participant in the younger placebo group experienced a Grade 4 SAEof unevaluable event (unknown of unknown origin [no additionalinformation currently available at the time of this report) 8 days afterDose 1 and died the same day.

One participant in the older placebo group experienced a Grade 4 SAE ofhemorrhagic stroke 15 days after Dose 2 and died 35 days after Dose 2.

Death Narratives

Narratives for the participants who died through the data cutoff date (6Oct. 2020) were provided.

Serious Adverse Events—Phase 2/3 First 6610 Participants—Phase 2/3

From Dose 1 to 1 month after Dose 2, the number of participants whoreported at least 1 SAE was similar in the BNT162b2 group (18 (0.5%])and in the placebo group (17 [0.5%]) (Table 16). None of the SAEs wereassessed by the investigator as related to study intervention. Most PTsfor SAEs were reported by only 1 participant (3 participants reported anSAE of appendicitis).

From Dose 1 to 1 month after Dose 2, the number of participants whoreported at least 1 SAE in the younger and older age groups was similar.

In the BNT162b2 group:

Two participants had an SAE each of appendicitis: 1 event began 9 daysafter Dose 1 and the other event began 15 days after Dose 2.

One participant had an SAE each of appendicitis and peritoneal abscess 7days after Dose 1, which was considered life-threatening. Both eventslasted for 17 days.

One participant had 8 SAEs 17 days after Dose 1: anemia, cardiac failurecongestive, abdominal adhesions, sepsis, hypokalaemia, mental statuschanges, acute kidney injury, and acute respiratory failure (all weresevere). The SAEs of abdominal adhesions and acute respiratory failurelasted for 2 and 14 days, respectively. All other SAEs lasted for 19days.

One participant had an SAE of anaphylactic reaction 9 days after Dose 2as a result of a bee sting which was considered life threatening. Theevent resolved on the same day.

In the placebo group, 1 participant had an SAE each of appendicitisperforated and peritonitis 13 and 15 days after Dose 2, respectively(both severe). Both events lasted 4 and 5 days, respectively.

From 1 month after Dose 2 to the data cutoff date, no additional SAEswere reported for these first 6610 participants.

TABLE 16 Number (%) of Subjects Reporting at Least 1 Serious AdverseEvent From Dose 1 to 1 Month After Dose 2, by System Organ Class andPreferred Term - ~6000 Subjects for Phase 2/3 Analysis - SafetyPopulation Vaccine Group (as Administered) BNT162b2 (30 μg) PlaceboSystem Organ Class (N^(a) = 3314) (N^(a) = 3296) Preferred Term n^(b)(%) (95% CI^(c)) n^(b) (%) (95% CI^(c)) Any event 18 (0.5)  (0.3, 0.9)17 (0.5)  (0.3, 0.8) BLOOD AND LYMPHATIC SYSTEM 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) DISORDERS Anaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) CARDIACDISORDERS 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Acute coronary syndrome0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Atrial fibrillation 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Cardiac failure congestive 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Coronary artery disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary arterydissection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery occlusion 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) EAR AND LABYRINTH DISORDERS 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Vertigo 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) EYE DISORDERS1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diplopia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)GASTROINTESTINAL DISORDERS 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3)Abdominal adhesions 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Colitis 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Diarrhoea 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Diverticular perforation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Intestinalobstruction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Small intestinal obstruction0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) GENERAL DISORDERS AND 2 (0.1) (0.0, 0.2)1 (0.0) (0.0, 0.2) ADMINISTRATION SITE CONDITIONS Chest pain 0 (0.0,0.1) 1 (0.0) (0.0, 0.2) Non-cardiac chest pain 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) Unevaluable event 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vascularstent occlusion 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) HEPATOBILIARY DISORDERS2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Cholelithiasis 2 (0.1) (0.0, 0.2)0 (0.0, 0.1) Cholecystitis acute 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) IMMUNESYSTEM DISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Anaphylactic reaction 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) INFECTIONS AND INFESTATIONS 4 (0.1) (0.0,0.3) 3 (0.1) (0.0, 0.3) Appendicitis 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1)Appendicitis perforated 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Peritonealabscess 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Peritonitis 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) Pneumonia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sepsis 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) Urinary tract infection 0 (0.0, 0.1) 1 (0.0)(0.0, 0.2) INJURY, POISONING AND 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2)PROCEDURAL COMPLICATIONS Forearm fracture 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Skin laceration 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INVESTIGATIONS 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Cardiac stress test abnormal 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) METABOLISM AND NUTRITION 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) DISORDERS Hypokalaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMSBENIGN, 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) MALIGNANT AND UNSPECIFIED(INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) Breast cancer 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaginalneoplasm 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEM DISORDERS 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Syncope 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)PSYCHIATRIC DISORDERS 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Bipolardisorder 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Mental disorder 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Mental status changes 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)Suicidal ideation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) RENAL AND URINARY 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) DISORDERS Acute kidney injury 1 (0.0)(0.0, 0.2) 0 (0.0, 0.1) RESPIRATORY, THORACIC 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) AND MEDIASTINAL DISORDERS Acute respiratory failure 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) VASCULAR DISORDERS 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2)Deep vein thrombosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Orthostatichypotension 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Note: MedDRA (v23.0) codingdictionary applied. ^(a)N = number of subjects in the specified group.This value is the denominator for the percentage calculations. ^(b)n =Number of subjects reporting at least 1 occurrence of the specifiedevent. For “any event”, n = number of subjects reporting at least 1occurrence of any event. ^(c)Exact 2-sided CI based on the Clopper andPearson method.

All Participants—Phase 2/3

From Dose 1 to the data cutoff date, inclusive of those discussed forthe first 6610 participants, the number of participants who reported atleast 1 SAE was similar in the BNT162b2 group (63 [0.3%]) and in theplacebo group (49 [0.3%]) (Table 17). Additional events included:

In the BNT162b2 group, there were 2 participants in the younger agegroup with an SAE each assessed by the investigator as related to studyintervention:

One participant had an SAE of lymphadenopathy (right axilla) 13 daysafter Dose 1, which was not resolved at the time of the data cutoff. Theparticipant was a 48-year-old woman with a relevant medical history ofeczema and topical crisaborole use who was administered BNT162b2 vaccinein the left deltoid and had right axillary pain and lymphadenopathy. Shehad no injuries to the right arm, no fever, and no history of a similarincident. Her WBC was normal with a normal lymphocyte count and a rightaxilla ultrasound showed 4 enlarged lymph nodes (largest 2.5×1.1×2.4cm). A biopsy was performed and was reported to be normal and withoutmarkers for lymphoma or other cancer. A follow-up visit with oncology(and possible repeat ultrasound) was planned for 3 months time.

One participant had an SAE of shoulder injury related to vaccineadministration (SIRVA, erroneously administered into or near theshoulder joint capsule) after Dose 2, which was recovering at the timeof the data cutoff.

From Dose 1 to the data cutoff date, a total of 6 participants in theBNT162b2 group reported an SAE of appendicitis. Three of these SAEs ofappendicitis occurred in the first 6610 participants. The 3 additionalSAEs of appendicitis are described below along with other specified SAEsthat were assessed as not related to study intervention in the BNT162b2group:

Two participants had an SAE each of appendicitis: 1 event began 17 daysafter Dose 1 which lasted for 3 days (younger age group), and the otherevent began 11 days after Dose 1 which lasted 5 days (older age group).

One participant in the older age group had an SAE of appendicitisperforated on the same day after Dose 1, which was resolving at the timeof the data cutoff.

One participant in the younger age group had an SAE of suspectedCOVID-19 on the same day after Dose 2, which lasted for 6 days. Thenasal swab result was negative.

TABLE 17 Number (%) of Subjects Reporting at Least 1 Serious AdverseEvent From Dose 1 to Data Cutoff Date (6 OCT. 2020), by System OrganClass and Preferred Term - Phase 2/3 (All Participants) - SafetyPopulation Vaccine Group (as Administered) BNT162b2 (30 μg) PlaceboSystem Organ Class (N^(a) = 18419) (N^(a) = 18436) Preferred Term n^(b)(%) (95% CI^(c)) n^(b) (%) (95% CI^(c)) Any event 63 (0.3)  (0.3, 0.4)49 (0.3)  (0.2, 0.4) BLOOD AND LYMPHATIC 2 (0.0) (0.0, 0.0) 2 (0.0)(0.0, 0.0) SYSTEM DISORDERS Anaemia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Leukocytosis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Lymphadenopathy 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) Neutropenia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0)Thrombocytosis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) CARDIAC DISORDERS 10(0.1)  (0.0, 0.1) 6 (0.0) (0.0, 0.1) Cardiac failure congestive 2 (0.0)(0.0, 0.0) 1 (0.0) (0.0, 0.0) Acute coronary syndrome 1 (0.0) (0.0, 0.0)1 (0.0) (0.0, 0.0) Atrial fibrillation 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0,0.0) Acute myocardial infarction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Anginapectoris 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Arrhythmia 0 (0.0, 0.0) 1 (0.0)(0.0, 0.0) Arrhythmia supraventricular 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Arteriospasm coronary 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Coronary arterydisease 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Coronary artery dissection 1(0.0) (0.0, 0.0) 0 (0.0, 0.0) Coronary artery occlusion 0 (0.0, 0.0) 1(0.0) (0.0, 0.0) Myocardial infarction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)EAR AND LABYRINTH DISORDERS 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Vertigo 1(0.0) (0.0, 0.0) 0 (0.0, 0.0) EYE DISORDERS 1 (0.0) (0.0, 0.0) 1 (0.0)(0.0, 0.0) Diplopia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Retinal arteryocclusion 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) GASTROINTESTINAL DISORDERS 7(0.0) (0.0. 0.1) 5 (0.0) (0.0, 0.1) Small intestinal obstruction 1 (0.0)(0.0, 0.0) 1 (0.0) (0.0, 0.0) Abdominal adhesions 1 (0.0) (0.0, 0.0) 0(0.0, 0.0) Colitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Diarrhoea 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) Diverticular perforation 0 (0.0, 0.0) 1 (0.0)(0.0, 0.0) Gastrointestinal haemorrhage 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Intestinal obstruction 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Obstructivepancreatitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Oesophageal food impaction0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Pancreatitis 1 (0.0) (0.0, 0.0) 0 (0.0,0.0) Salivary gland calculus 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) GENERALDISORDERS AND 3 (0.0) (0.0, 0.0) 3 (0.0) (0.0, 0.0) ADMINISTRATION SITECONDITIONS Unevaluable event 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Chest pain0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Influenza like illness 0 (0.0, 0.0) 1(0.0) (0.0, 0.0) Non-cardiac chest pain 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Shoulder injury related to vaccine 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)administration Vascular stent occlusion 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)HEPATOBILIARY DISORDERS 3 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0)Cholecystitis acute 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Cholelithiasis 2(0.0) (0.0, 0.0) 0 (0.0, 0.0) Bile duct stone 1 (0.0) (0.0, 0.0) 0 (0.0,0.0) IMMUNE SYSTEM DISORDERS 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Anaphylactic reaction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) INFECTIONS ANDINFESTATIONS 15 (0.1)  (0.0, 0.1) 9 (0.0) (0.0, 0.1) Appendicitis 5(0.0) (0.0, 0.1) 0 (0.0, 0.0) Pneumonia 1 (0.0) (0.0, 0.0) 4 (0.0) (0.0,0.1) Appendicitis perforated 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0)Cellulitis 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Diverticulitis 2 (0.0)(0.0, 0.0) 0 (0.0, 0.0) Pyelonephritis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Osteomyelitis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Peritoneal abscess 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) Peritonitis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0)Pharyngitis streptococcal 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Sepsis 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) Suspected COVID-19 1 (0.0) (0.0, 0.0) 0 (0.0,0.0) Urinary tract infection 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Urosepsis 0(0.0, 0.0) 1 (0.0) (0.0, 0.0) INJURY, POISONING AND PROCEDURAL 2 (0.0)(0.0, 0.0) 2 (0.0) (0.0, 0.0) COMPLICATIONS Forearm fracture 0 (0.0,0.0) 1 (0.0) (0.0, 0.0) Head injury 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hipfracture 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Skin laceration 0 (0.0, 0.0) 1(0.0) (0.0, 0.0) INVESTIGATIONS 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0)Cardiac stress test abnormal 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hepaticenzyme increased 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) METABOLISM ANDNUTRITION 3 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) DISORDERS Fluidretention 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hyperglycaemia 1 (0.0) (0.0,0.0) 0 (0.0, 0.0) Hypoglycaemia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0)Hypokalaemia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) MUSCULOSKELETAL AND 1 (0.0)(0.0, 0.0) 2 (0.0) (0.0, 0.0) CONNECTIVE TISSUE DISORDERSMusculoskeletal chest pain 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0)Osteoarthritis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Osteochondritis 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) NEOPLASMS BENIGN, MALIGNANT 3 (0.0) (0.0, 0.0) 2(0.0) (0.0, 0.0) AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinomagastric 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Breast cancer 0 (0.0, 0.0) 1(0.0) (0.0, 0.0) Metastases to central nervous 1 (0.0) (0.0, 0.0) 0(0.0, 0.0) system Uterine leiomyoma 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0)Vaginal neoplasm 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) NERVOUS SYSTEMDISORDERS 5 (0.0) (0.0, 0.1) 7 (0.0) (0.0, 0.1) Syncope 0 (0.0, 0.0) 3(0.0) (0.0, 0.0) Subarachnoid haemorrhage 2 (0.0) (0.0, 0.0) 0 (0.0,0.0) Cerebrovascular accident 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Diplegia 0(0.0, 0.0) 1 (0.0) (0.0, 0.0) Haemorrhagic stroke 0 (0.0, 0.0) 1 (0.0)(0.0, 0.0) Ischaemic stroke 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Loss ofconsciousness 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Paraesthesia 0 (0.0, 0.0)1 (0.0) (0.0, 0.0) Transient ischaemic attack 1 (0.0) (0.0, 0.0) 0 (0.0,0.0) PREGNANCY, PUERPERIUM AND 0 (0.0, 0.0) 3 (0.0) (0.0, 0.0) PERINATALCONDITIONS Pregnancy 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Abortionspontaneous incomplete 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) PSYCHIATRICDISORDERS 3 (0.0) (0.0, 0.0) 3 (0.0) (0.0, 0.0) Suicidal ideation 0(0.0, 0.0) 2 (0.0) (0.0, 0.0) Bipolar disorder 0 (0.0, 0.0) 1 (0.0)(0.0, 0.0) Mental disorder 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Mental statuschanges 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Psychotic disorder 1 (0.0) (0.0,0.0) 0 (0.0, 0.0) RENAL AND URINARY DISORDERS 4 (0.0) (0.0, 0.1) 0 (0.0,0.0) Nephrolithiasis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Acute kidney injury1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Renal colic 1 (0.0) (0.0, 0.0) 0 (0.0,0.0) RESPIRATORY, THORACIC AND 2 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0)MEDIASTINAL DISORDERS Acute respiratory failure 1 (0.0) (0.0, 0.0) 0(0.0, 0.0) Pneumonitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Pulmonaryembolism 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) SURGICAL AND MEDICAL 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) PROCEDURES Breast conserving surgery 1 (0.0)(0.0, 0.0) 0 (0.0, 0.0) UNCODED TERM 3 (0.0) (0.0, 0.0) 1 (0.0) (0.0,0.0) INVASIVE DUCTAL CARCINOMA 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) STAGE 1B,LEFT BREAST@@ LEFT OVARIAN CYST, BENIGN 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)TUMOR@@ MRSA INFECTION RIGHT 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) STUMP@@PROLAPSED UTERUS@@ 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) STEMI: ST ELEVATION 1(0.0) (0.0, 0.0) 0 (0.0, 0.0) MYOCARDIAL INFARCTION@@ VASCULAR DISORDERS4 (0.0) (0.0, 0.1) 2 (0.0) (0.0, 0.0) Deep vein thrombosis 1 (0.0) (0.0,0.0) 1 (0.0) (0.0, 0.0) Arteriosclerosis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0)Hypertension 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hypertensive urgency 1(0.0) (0.0, 0.0) 0 (0.0, 0.0) Orthostatic hypotension 0 (0.0, 0.0) 1(0.0) (0.0, 0.0) Note: MedDRA (v23.0) coding dictionary applied. ^(a)N =number of subjects in the specified group. This value is the denominatorfor the percentage calculations. ^(b)n = Number of subjects reporting atleast 1 occurrence of the specified event. For “any event”, n = numberof subjects reporting at least 1 occurrence of any event. ^(c)Exact2-sided CI based on the Clopper and Pearson method.

Serious Adverse Event Narratives—Phase 2/3

Narratives for the Phase 3 participants who reported SAEs assessed asrelated to study intervention by the investigator who completed theirvisit at 1 month after Dose 2 and through the data cutoff date (6 Oct.2020) were provided.

Safety-Related Participant Withdrawals—Phase 2/3 First 6610Participants—Phase 2/3

From Dose 1 to 1 month after Dose 2, 6 (0.2%) participants in theBNT162b2 group and 5 (0.2%) participants in the placebo group werewithdrawn because of AEs (Table 18), and no additional withdrawals werereported for these participants from 1 month after Dose 2 until the datacutoff date.

Withdrawals of interest in the BNT162b2 group:

Two participants were withdrawn because of AEs that were assessed by theinvestigator as related to study intervention. One participant in theyounger age group had an AE of myalgia 8 days after Dose 1 which wasrecovering at the time of the data cutoff. One participant in the olderage group had an AE of pruritus and an AE of tachycardia 2 days afterDose 1; both events had a duration of 1 day and both were severe.

Three participants each had an SAE and were withdrawn from the study:younger age group (gastric adenocarcinoma), and in the older age group(coronary artery disease and coronary artery dissection).

Withdrawals of interest in the placebo group:

One participant (younger age group) was withdrawn because of an AE ofallergy to vaccine (study intervention) and an AE of erythematous rash 2days after Dose 1; both AEs resolved 18 days later, and both wereassessed by the investigator as related to study intervention.

One participant in the older group had an SAE (coronary arteryocclusion) assessed by the investigator as not related and was withdrawnfrom the study.

One participant in the older group was withdrawn from the study becauseof an AE of urticaria 10 days after Dose 1. The event resolved on thesame day and was assessed by the investigator as not related to studyintervention.

TABLE 18 Number (%) of Subjects Withdrawn Because of Adverse Events FromDose 1 to 1 Month After Dose 2, by System Organ Class and PreferredTerm - ~6000 Subjects for Phase 2/3 Analysis - Safety Population VaccineGroup (as Administered) BNT162b2 (30 μg) Placebo System Organ Class(N^(a) = 3314) (N^(a) = 3296) Preferred Term n^(b) (%) (95% CI^(c))n^(b) (%) (95% CI^(c)) Any event 6 (0.2) (0.1, 0.4) 5 (0.2) (0.0, 0.4)CARDIAC DISORDERS 3 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) Atrialfibrillation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Coronary artery disease 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery dissection 1 (0.0) (0.0,0.2) 0 (0.0, 0.1) Coronary artery occlusion 0 (0.0, 0.1) 1 (0.0) (0.0,0.2) Left ventricular hypertrophy 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Tachycardia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GASTROINTESTINAL DISORDERS 0(0.0, 0.1) 1 (0.0) (0.0, 0.2) Diverticular perforation 0 (0.0, 0.1) 1(0.0) (0.0, 0.2) IMMUNE SYSTEM DISORDERS 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2)Allergy to vaccine 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INJURY, POISONING AND1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) PROCEDURAL COMPLICATIONS Ankle fracture1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fall 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1)MUSCULOSKELETAL AND 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) CONNECTIVE TISSUEDISORDERS Muscular weakness 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Myalgia 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMS BENIGN, 1 (0.0) (0.0, 0.2) 0(0.0, 0.1) MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS)Adenocarcinoma gastric 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEMDISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Transient ischaemic attack 1(0.0) (0.0, 0.2) 0 (0.0, 0.1) SKIN AND SUBCUTANEOUS 1 (0.0) (0.0, 0.2) 2(0.1) (0.0, 0.2) TISSUE DISORDERS Pruritus 1 (0.0) (0.0, 0.2) 0 (0.0,0.1) Rash erythematous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Urticaria 0 (0.0,0.1) 1 (0.0) (0.0, 0.2) Note: MedDRA (v23.0) coding dictionary applied.^(a)N = number of subjects in the specified group. This value is thedenominator for the percentage calculations. ^(b)n = Number of subjectsreporting at least 1 occurrence of the specified event. For “any event”,n = number of subjects reporting at least 1 occurrence of any event.^(c)Exact 2-sided CI based on the Clopper and Pearson method.

All Participants—Phase 2/3

From Dose 1 to the data cutoff date, 18 (0.1%) participants in theBNT162b2 group and 14 (0.1%) participants in the placebo group werewithdrawn because of AEs. In addition to withdrawals discussed for thefirst 6610 participants, other withdrawals included:

One participant in the younger BNT162b2 group had an SAE oflymphadenopathy (right axilla) 13 days after Dose 1 assessed by theinvestigator as related to study intervention and was withdrawn, whichwas not resolved at the time of the data cutoff.

Three participants in the younger age group (1 BNT162b2 and 2 placebo)became pregnant after Dose 1 and were withdrawn.

One participant in the younger placebo group had a positive pregnancytest (exposure during pregnancy) 39 days after Dose 1 and was withdrawn.

Narratives of Safety-Related Participant Withdrawals—Phase 2/3

Narratives for the Phase 2/3 participants with any AEs leading towithdrawal from the study through the data cutoff date (6 Oct. 2020)were provided.

Other Significant Adverse Events—Phase 2/3

AEs of special interest were not defined for Phase 2/3 of this study;however, targeted medical events were monitored throughout the study.

Other Safety Assessments—Phase 2/3 Severe COVID-19 Illness—Phase 2/3

At the time of the efficacy interim analysis cutoff date of 4 Nov. 2020,all 7 severe COVID-19 cases were reported in the placebo group.

Pregnancy—Phase 2/3

Pregnancy was reported in 5 Phase 3 participants through the data cutoffdate of 6 Oct. 2020: in 1 participant in the BNT162b2 group and 4participants in the placebo group. Incomplete spontaneous abortionoccurred in 1 participant in the placebo group.

Narratives for pregnancy were provided.

Analysis and Discussion of Deaths, Serious Adverse Events,Safety-Related Participant Withdrawals, and Other Significant AdverseEvents—Phase 2/3

Up to the data cutoff date of 6 Oct. 2020, the numbers of SAEs weresimilar in the BNT162b2 group (63 [0.3%]) and in the placebo group (49[0.3%]). Two participants in the BNT162b2 group reported SAEs that wereassessed by the investigator as related to study intervention.

Few participants in the BNT162b2 group (18 [0.1%]) and in the placebogroup (14 [0.1%]) were withdrawn because of AEs.

There were 3 deaths (1 in the BNT162b2 group and 2 in the placebogroup); none of the deaths were assessed by the investigator as relatedto study intervention.

Phase 2/3 Safety Conclusions

Across age groups, local reactions were generally similar in frequencyafter each dose, and systemic events generally increased in frequencyand severity after Dose 2 compared to Dose 1. Local and systemicreactogenicity events were well-tolerated and short-lived (mediandurations of 1.0 to 2.0 days).

Reactogenicity events after each dose of BNT162b2 in older adults weregenerally milder and less frequent than those observed in youngeradults. The majority of reactogenicity events were mild or moderate inseverity. No Grade 4 events were reported other than fever in 1participant in the BNT162b2 group that began 1 day after Dose 2 andlasted 1 day.

The reactogenicity and AE profile after BNT162b2 30 μg evaluated in 6610participants was consistent with the safety profile observed afterBNT162b2 30 μg in Phase 1 and Phase 2.

AEs were reported in 16.8% of participants in the BNT162b2 group, andmost AEs were mild or moderate in severity. At the time of the datacutoff date, the number of participants with AEs in the BNT162b2 groupwas greater as compared with the placebo group (7.9%), which uponanalysis, was attributed to reactogenicity events reported as AEs within7 days after each dose.

At the time of the data cutoff date, there were 2 related SAEs in theBNT162b2 group (lymphadenopathy and shoulder injury related to vaccineadministration (SIRVA, erroneously administered into or near theshoulder joint capsule), and there were 6 discontinuations because ofrelated AEs. There was 1 death in the BNT162b2 group (arteriosclerosis)and 2 deaths in the placebo group that were assessed as not related tostudy intervention.

Overall, BNT162b2 at 30 μg was well tolerated when measured up to 1month after Dose 2 following dosing regimen.

Example 16: Conclusion of Phase 3 Study of COVID-19 Vaccine, Meeting allPrimary Efficacy Endpoints

After conducting the final efficacy analysis in the ongoing Phase 3study, the mRNA-based COVID-19 vaccine, BNT162b2, met all of the study'sprimary efficacy endpoints. Analysis of the data indicated a vaccineefficacy rate of 95% (p<0.0001) in participants without prior SARS-CoV-2infection (first primary objective) and also in participants with andwithout prior SARS-CoV-2 infection (second primary objective), in eachcase measured from 28 days after the first dose, 7 days after the seconddose. The first primary objective analysis is based on 170 cases ofCOVID-19 of which 162 cases of COVID-19 were observed in the placebogroup versus 8 cases in the BNT162b2 group. Efficacy was consistentacross age, gender, race and ethnicity demographics. The observedefficacy in adults over 65 years of age was over 94%.

There were 10 severe cases of COVID-19 observed in the trial, with nineof the cases occurring in the placebo group and one in the BNT162b2vaccinated group. No serious safety concerns related to the vaccine werereported. A review of unblinded reactogenicity data from the finalanalysis which consisted of a randomized subset of at least 8,000participants 18 years and older in the Phase 2/3 study demonstrated thatthe vaccine was well tolerated, with most solicited adverse eventsresolving shortly after vaccination. The only Grade 3 (severe) solicitedadverse events greater than or equal to 2% in frequency after the firstor second dose were fatigue at 3.8% and headache at 2.0% following dose2. Consistent with earlier shared results, older adults tended to reportfewer and milder solicited adverse events following vaccination. Thelocal reactogenicity profile among SARS-CoV-2 positive participants wasconsistent with that of the overall reactogenicity subset; similarly, oncomparison of AE data with that of the ‘All subjects’, there is noindication of a worse safety profile in baseline positive participants.Indeed, there is no indication of a worse safety profile in baselinepositive participants; therefore, BNT162b2 can be used irrespective ofCOVID-19 history or SARS-CoV-2 serological status.

In addition, the safety milestone required by the U.S. Food and DrugAdministration (FDA) for Emergency Use Authorization (EUA) has beenachieved.

The results of this first global trial to reach the final efficacyanalysis mark indicated that a high rate of protection against COVID-19can be achieved very fast after the first 30 μg dose, underscoring thepotential of BNT162 to provide early protection.

In summary:

-   -   Primary efficacy analysis demonstrated BNT162b2 to be 95%        effective against COVID-19 beginning 28 days after the first        dose; 170 confirmed cases of COVID-19 were evaluated, with 162        observed in the placebo group versus 8 in the vaccine group    -   Efficacy was consistent across age, gender, race and ethnicity        demographics; observed efficacy in adults over 65 years of age        was over 94%    -   Safety data milestone required by U.S. Food and Drug        Administration (FDA) for Emergency Use Authorization (EUA) has        been achieved    -   Data demonstrates vaccine was well tolerated across all        populations with over 43,000 participants enrolled; no serious        safety concerns observed; the only Grade 3 adverse event greater        than 2% in frequency was fatigue at 3.8% and headache at 2.0%

Example 17: All Confirmed Cases of COVID-19 after Dose 1

A number of confirmed cases of COVID-19 are not captured in the analysesof the first primary endpoint for the evaluable efficacy populationbecause they occurred less than 7 days after Dose 2, or because theyoccurred in participants who were excluded from the evaluable efficacypopulation or who had evidence of infection before or during thevaccination regimen.

All reports of COVID-19 with onset at any time after Dose 1 areaccounted for in Table 19, which provides a summary of cases for allparticipants in the Dose 1 all-available efficacy (modifiedintention-to-treat) population, regardless of evidence of infectionbefore or during the vaccination regimen. Among these participants, 50cases of COVID-19 occurred after Dose 1 in the BNT162b2 group comparedto 275 cases in the placebo group (Table 19). Notably, in the BNT162b2group, most cases occurred before Dose 2. The estimated VE againstconfirmed COVID-19 occurring after Dose 1 was 82% (2-sided 95% CI:75.6%, 86.9%), with an estimated VE of 52.4% (2-sided 95% CI: 29.5%,68.4%) against confirmed COVID-19 occurring after Dose 1 but before Dose2.

TABLE 19 COVID-19 Occurrence After Dose 1 - Dose 1 All-AvailableEfficacy Population BNT162b2 (30 μg) Placebo (N^(a) = 21669) (N^(a) =21686) Efficacy Endpoint n^(b) n^(b) COVID-19 occurrence after Dose 1 50275 After Dose 1 to before Dose 2 39 82 Dose 2 to 7 days after Dose 2 221 ≥7 days after Dose 2 9 172 ^(a)N = number of subjects in thespecified group. ^(b)n1 = Number of subjects meeting the endpointdefinition.

The early onset of protection is readily apparent in FIG. 100 , whichdisplays cumulative incidence for the first COVID-19 occurrence afterDose 1 among all vaccinated participants based on Dose 1 all-availableefficacy (modified intention-to-treat) population. Disease onset appearsto track together for BNT162b2 and placebo until approximately 14 daysafter Dose 1, at which point the curves diverge, with cases steadilyaccumulating in the placebo group, while remaining virtually flat in theBNT162b2 group.

The cumulative incidence of COVID-19 cases over time among placebo andvaccine recipients begins to diverge by 14 days after Dose 1,approximately 9 days after the estimated median incubation period of 5days, indicating the early onset of a partially protective effect ofimmunization. In the interval between Dose 1 and Dose 2, the observedvaccine efficacy was 52%, and in the first 7 days after Dose 2, it was91%, reaching full efficacy for COVID-19 with onset at least 7 daysafter Dose 2.

Example 18: Secondary Efficacy Results—Final Analysis

Vaccine Efficacy for COVID-19 Occurring within 14 Days after Dose2—Final AnalysisParticipants without Evidence of Infection Before Vaccination

For this efficacy endpoint, participants with positive or unknown NAATresults at any illness visit prior to 14 days after Dose 2 were notincluded in the evaluation for efficacy.

Among participants without evidence of SARS-CoV-2 infection before andduring vaccination regimen, VE against confirmed COVID-19 occurring atleast 14 days after Dose 2 was 94.2%, with 8 and 139 cases in theBNT162b2 and placebo groups respectively. The posterior probabilityof >99.99% for the true VE greater than 30% met the prespecified successcriterion of >98.6% for this endpoint. The 95% credible interval for thevaccine efficacy was 88.7% to 97.2%, indicating that the true VE is atleast 88.7% with a 97.5% probability given the available data.

Participants with or without Evidence of Infection Before Vaccination

Among participants with or without evidence of SARS-CoV-2 infectionbefore and during vaccination regimen, VE against confirmed COVID-19occurring at least 14 days after Dose 2 was 94.4%, with 8 and 144 casesin the BNT162b2 and placebo groups respectively. The posteriorprobability of >99.99% for the true VE greater than 30% met theprespecified success criterion of >98.6% for this endpoint. The 95%credible interval for the vaccine efficacy was 89.1% to 97.3%,indicating that the true VE is at least 89.1% with a 97.5% probabilitygiven the available data.

Vaccine Efficacy for Severe COVID-19 Cases—Final Analysis

Efficacy Against Severe COVID-19 (≥7 Days after Dose 2)Participants without Evidence of Infection Before and During VaccinationRegimen

For this efficacy endpoint, participants with positive or unknown NAATresults at any illness visit prior to 7 days after Dose 2 were notincluded in the evaluation for efficacy. Among participants withoutevidence of severe SARS-CoV-2 infection before and during vaccinationregimen, the estimated VE against severe COVID-19 occurring at least 7days after Dose 2 was 66.4%, with 1 and 3 cases in the BNT162b2 andplacebo groups respectively. The posterior probability for the truevaccine efficacy greater than 30% is 74.29%, which did not meet theprespecified success criterion of >98.6% for this endpoint due to thesmall number of severe cases observed after Dose 2 in the study.

Participants with and without Evidence of Infection Before and DuringVaccination Regimen

Among participants with or without evidence of severe SARS-CoV-2infection prior to 7 days after Dose 2, VE against severe COVID-19occurring at least 7 days after Dose 2 was 66.3%, with 1 and 3 cases inthe BNT162b2 and placebo groups respectively. The posterior probabilityfor the true vaccine efficacy greater than 30% is 74.19%.

All Confirmed Cases of Severe COVID-19 after Dose 1—all-AvailablePopulation

Among participants in the all-available efficacy population, 1 case ofCOVID-19 occurred after Dose 1 in the BNT162b2 group compared to 9 casesin the placebo group. The estimated VE against severe COVID-19 occurringafter Dose 1 was 88.9% (2-sided 95% CI: 20.1%, 99.7%), with an estimatedVE of 75.0% against severe COVID-19 occurring at least 7 days after Dose2.

Efficacy Against Severe COVID-19 (214 Days after Dose 2)Participants without Evidence of Infection Before and During VaccinationRegimen (14 Days)—Severe

Among participants without evidence of severe SARS-CoV-2 infectionbefore and during vaccination regimen, the estimated VE against severeCOVID-19 occurring at least 14 days after Dose 2 was 66.4%, with 1 and 3cases in the BNT162b2 and placebo groups respectively. The posteriorprobability for the true vaccine efficacy greater than 30% is 74.32%.

Participants with or without Evidence of Infection Before and DuringVaccination Regimen (14 Days)—Severe

Among participants with or without evidence of severe SARS-CoV-2infection before and during vaccination phase, VE against severeCOVID-19 occurring at least 14 days after Dose 2 was 66.3%, with 1 and 3cases in the BNT162b2 and placebo groups respectively. The posteriorprobability for the true vaccine efficacy greater than 30% is 74.18%.

Vaccine Efficacy for COVID-19 Cases Per CDC Definition—Final Analysis

Efficacy Against COVID-19 Based on CDC-Defined Symptoms (≥7 Days afterDose 2)Participants without Evidence of Infection Before and During VaccinationRegimen—CDC Defined—7 Days

Among participants without evidence of SARS-CoV-2 infection before andduring vaccination regimen, VE against CDC-defined COVID-19 occurring atleast 7 days after Dose 2 was 95.1% (2-sided 95% CI: 90.2%, 97.9%), with8 and 165 cases in the BNT162b2 and placebo groups, respectively.

Participants with and without Evidence of Infection Before and DuringVaccination Regimen—CDC Defined—7 Days

Among participants with and without evidence of SARS-CoV-2 infectionbefore and during vaccination regimen, VE against CDC-defined COVID-19occurring at least 7 days after Dose 2 was 94.7% (2-sided 95% CI:89.8%-97.6%), with 9 and 172 cases in the BNT162b2 and placebo groups,respectively.

Efficacy Against COVID-19 Based on CDC-Defined Symptoms (≥14 Days afterDose 2)

Among participants without and with or without evidence of SARS-CoV-2infection before and during vaccination regimen, VE against CDC-definedCOVID-19 occurring at least 14 days after Dose 2 were similar to thoseoccurring at least 7 days after Dose 2.

Example 19: Efficacy Conclusions—Final Analysis

In the final efficacy analysis, among participants without evidence ofSARS-CoV-2 infection before and during vaccination regimen, VE againstconfirmed COVID-19 occurring at least 7 days after Dose 2 was 95.0%,with 8 COVID-19 cases in the BNT162b2 group compared to 162 COVID-19cases in the placebo group. The 95% credible interval for the vaccineefficacy was 90.3% to 97.6%. For the second primary endpoint, VE againstconfirmed COVID-19 occurring at least 7 days after Dose 2 inparticipants with and without evidence of SARS-CoV-2 infection beforeand during vaccination regimen was 94.6%, with 9 and 169 cases in theBNT162b2 and placebo groups respectively. The posterior probabilityof >99.99% for the true VE greater than 30% met the prespecified successcriterion of >98.6% for this endpoint. The 95% credible interval for thevaccine efficacy was 89.9% to 97.3%, indicating that the true VE is atleast 89.9% with a 97.5% probability given the available data

Observed VE was very high for the first primary efficacy endpoint acrosssubgroups of age, sex, race/ethnicity, and country, as VE was >93% inall subgroups, with the exception of “all others” race group (89.3% VE)and Brazil (87.7% VE).

A total of 10 cases of severe COVID-19 occurred after Dose 1, 1 in theBNT162b2 group, compared with 9 cases in the placebo group.

Among all participants (regardless of evidence of infection before orduring the vaccination regimen) 50 cases of COVID-19 occurred after Dose1 in the BNT162b2 group compared with 275 cases in the placebo group,indicating an estimated VE of 82% (95% CI: 75.6%, 86.9%) againstconfirmed COVID-19 occurring after Dose 1.

The early onset of protection is readily apparent from cumulativeincidence curves, which show that disease onset tracks conjointly forBNT162b2 and placebo until approximately 14 days after Dose 1, at whichpoint the curves diverge, with cases steadily accumulating in theplacebo group, while remaining virtually flat after BNT162b2.

In conclusion, the final efficacy results show that BNT162b2 at 30 μgprovided protection against COVID-19 in participants who had no evidenceof prior infection with SARS-CoV-2, including across demographicsubgroups, with severe cases observed predominantly in the placebogroup.

Details of demographic populations assessed are presented below inTables 20 and 21.

TABLE 20 Demographic Characteristics - ~38000 Subjects for Phase 2/3Analysis - Safety Population Vaccine Group (as Administered) BNT162b2(30 μg) Placebo Total (N^(a) = 18860) (N^(a) = 18846) (N^(a) = 37706)n^(b) (%) n^(b) (%) n^(b) (%) Sex Male 9639 (51.1) 9436 (50.1) 19075(50.6) Female 9221 (48.9) 9410 (49.9) 18631 (49.4) Race White 15636(82.9) 15630 (82.9) 31266 (82.9) Black or African American 1729 (9.2)1763 (9.4) 3492 (9.3) American Indian or Alaska 102 (0.5) 99 (0.5) 201(0.5) native Asian 801 (4.2) 807 (4.3) 1608 (4.3) Native Hawaiian orother 50 (0.3) 26 (0.1) 76 (0.2) Pacific Islander Multiracial 449 (2.4)406 (2.2) 855 (2.3) Not reported 93 (0.5) 115 (0.6) 208 (0.6) EthnicityHispanic/Latino 5266 (27.9) 5277 (28.0) 10543 (28.0)Non-Hispanic/non-Latino 13482 (71.5) 13459 (71.4) 26941 (71.5) Notreported 112 (0.6) 110 (0.6) 222 (0.6) Country Argentina 2883 (15.3)2881 (15.3) 5764 (15.3) Brazil 1145 (6.1) 1139 (6.0) 2284 (6.1) SouthAfrica 372 (2.0) 372 (2.0) 744 (2.0) USA 14460 (76.7) 14454 (76.7) 28914(76.7) Age group 16-55 Years 10889 (57.7) 10896 (57.8) 21785 (57.8) >55Years 7971 (42.3) 7950 (42.2) 15921 (42.2) Age at vaccination (years)Mean (SD) 50.5 (15.65) 50.3 (15.72) 50.4 (15.68) Median 52.0 52.0 52.0Min, max (16, 89) (16, 91) (16, 91) Body mass index (BMI) Underweight(<18.5 kg/m²) 201 (1.1) 235 (1.2) 436 (1.2) Normal weight (≥18.5 5517(29.3) 5460 (29.0) 10977 (29.1) kg/m²-24.9 kg/m²) Overweight (≥25.0kg/m²- 6578 (34.9) 6481 (34.4) 13059 (34.6) 29.9 kg/m²) Obese (≥30.0kg/m²) 6556 (34.8) 6662 (35.3) 13218 (35.1) Missing 8 (0.0) 8 (0.0) 16(0.0) Note: HIV-positive subjects are included in this summary but notincluded in the analyses of the overall study objectives. ^(a)N = numberof subjects in the specified group, or the total sample. This value isthe denominator for the percentage calculations. ^(b)n = Number ofsubjects with the specified characteristic.

TABLE 21 Baseline Charlson Comorbidities - ~38000 Subjects for Phase 2/3Analysis - Safety Population Vaccine Group (as Administered) BNT162b2(30 μg) Placebo Total Charlson Comorbidity (N^(a) = 18860) (N^(a) =18846) (N^(a) = 37706) Index Category n^(b) (%) n^(b) (%) n^(b) (%)Subjects with any 3934 (20.9) 3809 (20.2) 7743 (20.5) Charlsoncomorbidity AIDS/HIV 59 (0.3) 62 (0.3) 121 (0.3) Any Malignancy 733(3.9) 662 (3.5) 1395 (3.7) Cerebrovascular Disease 195 (1.0) 166 (0.9)361 (1.0) Chronic Pulmonary Disease 1478 (7.8) 1453 (7.7) 2931 (7.8)Congestive Heart Failure 88 (0.5) 83 (0.4) 171 (0.5) Dementia 7 (0.0) 11(0.1) 18 (0.0) Diabetes With Chronic 99 (0.5) 113 (0.6) 212 (0.6)Complication Diabetes Without Chronic 1473 (7.8) 1478 (7.8) 2951 (7.8)Complication Hemiplegia or Paraplegia 13 (0.1) 21 (0.1) 34 (0.1)Leukemia 12 (0.1) 10 (0.1) 22 (0.1) Lymphoma 22 (0.1) 32 (0.2) 54 (0.1)Metastatic Solid Tumor 4 (0.0) 3 (0.0) 7 (0.0) Mild Liver Disease 125(0.7) 89 (0.5) 214 (0.6) Moderate or Severe Liver 1 (0.0) 2 (0.0) 3(0.0) Disease Myocardial Infarction 194 (1.0) 188 (1.0) 382 (1.0) PepticUlcer Disease 52 (0.3) 71 (0.4) 123 (0.3) Peripheral Vascular 124 (0.7)117 (0.6) 241 (0.6) Disease Renal Disease 123 (0.7) 133 (0.7) 256 (0.7)Rheumatic Disease 62 (0.3) 56 (0.3) 118 (0.3) Note: MedDRA (v23.1)coding dictionary applied. Note: HIV-positive subjects are included inthis summary but not included in the analyses of the overall studyobjectives. ^(a)N = number of subjects in the specified group. Thisvalue is the denominator for the percentage calculations. ^(b)n = Numberof subjects with the specified characteristic. Subjects with multipleoccurrences within each category are counted only once. For ‘Subjectswith any Charlson comorbidity’, n = number of subjects reporting atleast 1 occurrence of any Charlson comorbidity.

Example 20: Certain Observations Regarding Response of Young AdolescentPopulations to Immunization with BNT162b2

In clinical trials described in Examples 13-19, the following wereobserved in younger adolescent populations.

Local Reactions in Younger Adolescents

Younger adolescents 12 to 15 years of age (N=100; 49 in the BNT162b2group and 51 in the placebo group) contributed preliminary data to thereactogenicity subset and were analyzed separately. In this age group,pain at the injection site was the most frequently prompted localreaction in the BNT162b2 group, reported in 71.4% of participantscompared to 17.6% in the placebo group after Dose 1. The incidence ofpain was reduced in the BNT162b2 group and placebo group after Dose 2(down to 58.7% vs 8.7%). Redness was reported in 1 participant in theBNT162b2 group after Dose 1 and in 2 participants after Dose 2, and innone in the placebo group after either dose. Swelling was reported in 2participants in the BNT162b2 group after Dose 1 and in 3 participantsafter Dose 2, and in 1 in the placebo group after Dose 1 and none afterDose 2. Most local reactions were mild to moderate in severity. Twosevere reactions were reported, both in the BNT162b2 group: severeredness and severe pain at the injection site.

Systemic Reactions in Younger Adolescents

Younger adolescents 12 to 15 years of age (N=100; 49 in the BNT162b2group and 51 in the placebo group) contributed preliminary data to thereactogenicity subset and were analyzed separately. Most systemic events(other than vomiting and diarrhea, which had low incidences acrossgroups) were reported at higher incidence in the BNT162b2 group than inthe placebo group. However, there was no clear trend for increasingincidence or severity after Dose 1 compared to after Dose 2. In this agegroup, the most frequent prompted systemic events after Dose 1 comparedto Dose 2 were (Dose 1 vs Dose 2):

-   -   fatigue: BNT162b2 (49.0% vs 50.0%) compared to placebo (25.5% vs        6.5%)    -   headache: BNT162b2 (42.9% vs 45.7%) compared to placebo (35.3%        vs 21.7%)    -   muscle pain: BNT162b2 (22.4% vs 30.4%) compared to placebo        (13.7% vs 4.3%)    -   chills: BNT162b2 (30.6% vs 28.3%) compared to placebo (7.8% vs        8.7%)    -   joint pain: BNT162b2 (12.2% vs 17.4%) compared to placebo (9.8%        vs 6.5%)    -   fever: BNT162b2 (14.3% vs 19.6%) compared to placebo (0% vs 0%)    -   vomiting: reported at similar frequencies in both groups and        similar after each dose    -   diarrhea: reported at similar frequencies in both groups and        similar after each dose.

Most systemic events in younger adolescents were mild to moderate inseverity. Severe events were relatively infrequent in both groups,occurring in no more than 1 or 2 participants after either dose.

Antipyretic/pain medication use in the younger adolescent group wasmodestly increased after Dose 2 compared to Dose 1 (30.6% vs 41.3%) andwas greater than use in the placebo group (9.8% vs 13%).

In summary, as observed in older age groups (e.g., greater than 16 yearsof age such as 16-85 years of age), reactogenicity was mostly mild tomoderate and short-lived after dosing for younger adolescents 12 to 15years of age, and the adverse event profile did not suggest any serioussafety concerns.

Examples 21-24 below further confirm that neutralizing antibodyresponses and/or cell-mediated immune responses can be achieved withmRNA compositions described herein (including, e.g., BNT162b1 andBNT162b2) administered according to various dosing regimens describedherein, including for example dosing regimens that involveadministration of one or more doses lower than 30 ug, including, e.g.,20 ug, 10 ug, 3 ug, etc. Among other things, data provided in theseExamples 21-24 further confirm induction of an immune response (e.g., asdescribed herein) against SARS-CoV-2 upon administration of certain mRNAcompositions described herein (including, e.g., BNT162b1 and BNT162b2)with one or more doses of 3 ug or above.

Those of ordinary skill in the art, reading the present disclosure, willappreciate that it demonstrates among other things, that administrationof various mRNA compositions described herein can induce immuneresponses that include neutralizing antibodies against SARS-CoV-2; itfurthermore confirms that certain such compositions (i.e., that induceneutralizing antibodies and/or that induce cell-mediated immune responsesuch as T cell response) can induce protective immune responses thatreduce SARS-CoV-2 infection and/or incidence of COVID19 sickness inorganisms, specifically including primate organisms in which they haveinduced such neutralizing antibodies and/or cell-mediated immuneresponse and furthermore including humans. In some embodiments, it alsoconfirms that certain such compositions (e.g., described herein) do notsignificantly induce vaccine-mediated disease enhancement, for example,as evidenced by only one of the 10 cases of severe COVID-19 that wereobserved after a first dose. Indeed, the present disclosure documentsthat such compositions can effectively vaccinate humans (see, forexample, clinical trial results included in Examples 13-19), forexample, against severe COVID-19 disease.

Example 21: Immunogenicity Studies for Functional Antibody Responses

In clinical trials described in Example 7, the following were observedin healthy younger adults (18-55 years of age) and older adults (56-85years of age) after BNT162b1 or BNT162b2 vaccination. Two doses, of 1μg, 3 μg, 10 μg, 20 μg, or 30 μg were administered 21 days apart inyounger adults. Two doses of 20 μg was administered 21 days apart inolder adults. Functional antibody data for younger adult cohorts wasdetermined up until Day 50 after an initial dose was administered fordose groups 1 μg and 3 μg, and up until Day 85 for dose groups 10, 20,and 30 μg. For BNT162b2-dosed older adults, data is available until Day29 after an initial dose was administered.

For virus neutralizing antibody GMTs (neutralizing GMTs) and 95%confidence intervals for participants aged 18 to 55 years after dosingwith BNT162b1, see FIG. 40 .

For virus neutralizing antibody GMTs (neutralizing GMTs) and 95%confidence intervals for younger participants aged 18 to 55 yrs andolder participants aged 56 to 85 yrs after dosing with BNT162b2, seeFIG. 101 (50% neutralizing titer).

Geometric means fold increase (GMFI) from baseline in functionalantibody titer data are shown in FIG. 102 (BNT162b1) and FIG. 103(BNT162b2).

Participants dosed with BNT162b1 showed a strong dose-dependent antibodyresponse. On Day 22, at 21 days after dose 1, virus neutralisingantibody GMTs had increased in a dose-dependent manner for the 1, 10, 30and 50 μg dose groups. At Day 29 (7 days after Dose 2), neutralisingGMTs showed a strong, dose level dependent booster response. In thesingle, 60 μg dose group, neutralising GMTs remained at a lower level,indicating a booster dose is necessary to increase functional antibodytiters.

On Day 43 (21 days after Dose 2 of BNT162b1), neutralising GMTsdecreased (with the exception of the 1 μg dose level). Day 43 virusneutralising GMTs were 0.7-fold (1 μg) to 3.6-fold (50 μg) those of aCOVID-19 HCS panel.

The COVID-19 HCS panel is comprised of 38 human COVID-19 HCS sera drawnfrom individuals aged 18 to 85 yrs, at least 14 d after confirmeddiagnosis, and at a time when the individuals were asymptomatic. Theserum donors predominantly had symptomatic infections (35/38), and onehad been hospitalized. The sera were obtained from Sanguine Biosciences(Sherman Oaks, Calif.), the MT Group (Van Nuys, Calif.), and PfizerOccupational Health and Wellness (Pearl River, N.Y.).

Participants dosed with BNT162b2 showed a strong antibody responseinduced by BNT162b2. Virus neutralizing GMTs were detected at 21 daysafter Dose 1 (Day 22) and had increased substantially in youngerparticipants (aged 18 to 55 years) immunized with ≥3 μg of BNT162b2, andolder participants (aged 56-85 years) immunized with 20 μg BNT162b2 by 7days after Dose 2 (Day 29). Day 29 virus neutralizing GMTs werecomparable between the younger and older adult 20 μg dose level cohorts.The lowest treated dose of 1 μg BNT162b2 elicited a minimal neutralisingresponse in participants aged 18 to 55 years.

On Day 43 (21 days after Dose two of BNT162b2), virus neutralising GMTsin the younger adult cohorts decreased for the 3, 20, and 30 μg doselevels. Thereafter, neutralising GMTs in between Days 29 and 43,neutralizing GMTs remained stable up to Day 85 (63 days after Dose two)for younger adult dose groups 10, 20 and 30 μg and were 1.3-fold to1.9-fold those of a COVID-19 HCS panel.

Seroconversion in this context is defined as a minimum of a 4-foldincrease of antibody GMT as compared to baseline. The frequency ofparticipants with seroconversion is shown in FIG. 104 (BNT162b1) and 105(BNT162b2).

All participants dosed with Dose 1 at ≥30 μg BNT162b1 or BNT162b2seroconverted either by 7 days or 21 days after Dose 2 (Day 29 or Day43). All participants dosed with 30 μg BNT162b2 remained seropositivethroughout the follow-up until Day 85.

Example 22: Immunogenicity Studies for Binding Antibody Concentrations

In clinical trials described in Example 7, the following were observedin healthy younger adults (18-55 years of age) and older adults (56-85years of age) after BNT162b1 or BNT162b2 vaccination. Binding antibodyconcentration data is available up until Day 43 for BNT162b1-dosedyounger participants aged 18 to 55 yrs dosed with 1, 10, 30, 50, or 60μg on Days 1 (all dose levels) and 22 (all dose levels except 60 μg)(n=12 per group).

For BNT162b2-dosed participants, data is available for youngerparticipants aged 18 to 55 yrs dosed with 1, 3, 10, 20, or 30 μg, andolder participants aged 56 to 85 yrs dosed with 20 μg on Days 1 and 22(n=12 per group). Binding antibody concentration data for youngerparticipant dose groups is available up until Day 50 for dose groups 1μg and 3 μg, and up until Day 85 for dose groups 10, 20, and 30 μg. Forthe BNT162b2-dosed older participants, data is available up until Day29.

The fold increase from baseline in binding antibody concentrations afterdosing with BNT162b1 and BNT162b2 are shown in FIG. 106 and FIG. 107 ,respectively. Participants dosed with BNT162b1 showed a strongdose-dependent antibody response against the SARS-CoV-2 spike (S)protein S1 subunit at Day 21 after Dose 1 (Day 22). At 7 days after Dose2 (Day 29), S1-binding immunoglobulin (IgG) GMCs showed a strong,dose-dependent booster response. In the 60 μg dose group, which was onlydosed once, S1-binding IgG GMCs remained at a lower level, indicatingthat a booster dose is necessary to increase antibody concentrations.

At 21 days after Dose 2 of BNT162b1 (Day 43), S1-binding IgG GMCsdecreased (with exception of the 1 μg dose group), but were clearlyabove those of a COVID-19 HSC panel for all doses tested.

BNT162b2 dosed participants showed a strong BNT162b2-induced S1-bindingIgG response at 21 days after Dose 1 (Day 22) with evidence of adose-dependent response only between the 1 μg and 10 μg dose levels.S1-binding IgG GMCs showed a substantial boster response by 7 days afterDose 2 (Day 29). Day 29 S1-binding IgG GMCs were comparable between theyounger and older participants at the 20 μg dose level.

Across all dose-level cohorts antibody levels decreased over time, butwith S1-binding antibody GMCs well above that observed in a COVID-19 HCSpanel at Day 85 (63 days after Dose 2; 10 to 30 μg dose level) (FIG. 107).

Almost all BNT162b1- and BNT162b2-immunized participants seroconvertedwith regard to the S1-binding antibody response as early as 21 daysafter Dose 1 (Day 22). Frequency of participants with seroconversionafter dosing with BNT162b1 is shown in FIG. 108 and with BNT162b2 isshown in FIG. 109 . Similar observations were made using only the RBDdomain as the target antigen.

Example 23: Exemplary Cell-Mediated Immune Responses:SARS-CoV-2-Specific CD4⁺ and CD8⁺ T-Cell Responses

In clinical trials described in Example 7, the following were observedin healthy younger adults (18-55 years of age) and older adults (56-85years of age) after BNT162b1 or BNT162b2 vaccination. CD4+ and CD8+T-cell response data were available from 97 study participants receivingBNT162b1, 70 younger participants at dose levels of 1, 3, 10, 20, 30,50, or 60 μg (note: Dose 2 was not given in the 60 μg dose group), and27 older participants at dose levels of 10, 20, or 30 μg, as well as 76participants receiving BNT162b2 at dose levels of 1, 3, 10, 20, or 30 μg(47 younger participants), or 10, 20, or 30 μg (older participants).

BNT162b1 induced strong RBD-specific CD4⁺ T-cell responses in themajority of participants given both dose one and dose two (86 of 88[97.7%]), including all older participants (27 of 27 [100%]); CD8⁺responses were induced in 47 of 61 (77.0%) younger participants and in21 of 27 (77.7%) of older participants. In contrast, T-cell responseswere detected less often and were lower in magnitude in 9 youngerparticipants who received only Dose 1 in the 60 μg dose group,indicating the importance of a booster dose

BNT162b2 induced strong SARS-CoV-2 S protein-specific CD4⁺ T-cellresponses in all of the dosed younger or older participants (76 of 76[100%]); CD8⁺ T-cell responses were induced in 45/47 (95.7%) of youngerparticipants and 24/29 (82.8%) older participants. Despite the slightlylower CD8⁺ immunogenicity rate in older participants, the magnitude ofthe BNT162b2-induced responses was comparable to those induced inyounger participants receiving 30 μg of BNT162b2. These T-cell responseswere directed against different parts of the antigen including non-RBDsequences, indicating the induction of multi-epitopic responses byBNT162b2 in both age groups.

Dosing twice with BNT162b1 or BNT162b2 led to a substantial increase inincidence and magnitude of T-cell responses in both age groups, andacross all dose levels for BNT162b1. While the magnitude of CD4⁺ T-cellresponses induced by BNT162b2 was also similar across different doselevels, the magnitude of CD8⁺ T-cell responses was highest at the 30 μgdose level. The participants with the strongest CD4⁺ T-cell responseshad more than 10-fold of the memory responses observed in the sameparticipants against immunodominant peptides from cytomegalovirus,Epstein Barr virus, influenza virus, and tetanus toxoid in the sameparticipants. The same participants also had strong CD8⁺ T-cellresponses that were comparable to memory responses against the abovementioned viral antigens.

RBD- and S protein-specific CD4⁺ T-cell responses observed aftervaccination were induced de novo by BNT162b1 in 97.5% of participantsand by BNT162b2 in 100% of participants. RBD- and S protein-specificCD8⁺ T-cell responses observed after vaccination were induced de novo byBNT162b1 in 95.5% of participants and by BNT162b2 in 96.6% ofparticipants.

Example 24: Exemplary Cell-Mediated Immune Responses: Functional andPro-Inflammatory CD4⁺/CD8⁺ T-Cell Responses

In clinical trials described in Example 7, the following were observedin healthy younger adults (18-55 years of age) and older adults (56-85years of age) after BNT162b1 or BNT162b2 vaccination. De novo inductionof SARS-CoV-2 S protein or RBD protein directed T-cells was confirmedusing intracellular cytokine staining (ICS). As described in Example 7for BNT162b1, similar cell-mediated immune responses were also observedwith BNT162b2 as described below.

For example, IFNγ-producing CD4+ and CD8+ T-cells against SARS-CoV-2 Sprotein or RBD were induced robustly by both BNT162b1 and BNT162b2. Noclear dose dependency was observed for both BNT162b1 and BNT162b2. Thecytokine responses elicited after dosing with either BNT162b1 orBNT162b2 in older participants was mostly identical in response patternand intensity with that in younger participants.

BNT162b1 and BNT162 induced poly-functional and pro-inflammatoryCD4+/CD8+ T-cell responses in almost all participants. The detection ofinterferon (IFN)γ, interleukin (IL)-2 but not IL-4 indicates a favorableTh1 profile and the absence of a potentially deleterious Th2 immuneresponse.

Regarding BNT162b2, peripheral blood mononuclear cell (PBMC) fractionsisolated from blood of study participants collected at baseline(pre-Dose 1) and 29±3 d after Dose 1 BNT162b2 were analyzed. Thisincludes data for a total of 74 study participants:

-   -   Younger participants aged 18 to 55 yrs per dose group: 1 μg        (n=8), 3 μg (n=9), 10 μg (n=10), 20 μg (n=9), 30 μg (n=10).    -   Older participants aged 56 to 85 yrs per dose group: 10 μg        (n=11), 20 μg (n=8), 30 μg (n=9).

The functionality and polarization of vaccine-induced SARS-CoV-2S-specific T cells were assessed by intracellular accumulation ofcytokines IFN-gamma, IL-2, and IL-4 in response to stimulation withoverlapping peptides representing the full-length sequence of thevaccine-encoded RBD and the wild-type SARS CoV-2 protein, respectively.As a control, PMBCs from 18 COVID-19 convalescent virologicallyconfirmed patients were used.

Two doses of BNT162b2 (dose range 1 to 30 μg), induced vaccine-specificT-cell responses in both age groups analyzed (FIGS. 110 and 111 ).Testing for SARS-CoV-2 S protein specific T-cell responses was performedwith two different peptide pools, S pool 1 comprising overlappingpeptides from the N-terminal region of the S protein (which is notequivalent to structural domains) and S pool 2 comprising C-terminalregions of the S protein. S-specific CD4⁺ T-cell responses analyzed in74 participants dosed with BNT162b2 were characterized by a Th1 cytokineprofile secreting IFN-gamma, or IL-2, or both.

Almost no Th2 cytokine IL-4 secreting T cells were detectable inresponse to S peptide sub-pool stimulations (mean fractions: 0.01% and0.02% of antigen-specific circulating CD4⁺ T cells in the 20 and 30 μgadult cohort, respectively; separate stimulation with S protein sub-pool1 and sub-pool 2). S-specific CD8⁺ T cells secreted IFNγ in 61 of the 74analyzed participants (adults: 40 of 46 participants and older adults:21 of 28 participants) and also IL-2 secreting CD8⁺ T cells weredetectable. Fractions of S-specific IFNγ⁺CD8⁺ T cells targeting theN-terminal domain of the S protein reached up to 1% of total peripheralblood CD8⁺ T cells in the 20 and 30 μg younger participant dose groupsand up to 2.4% in the 30 μg older participant dose group. Pre-existingCD8⁺ T-cell responses against the C-terminal region of the S proteinwere detected in 17 of 74 dosed participants (range: 0.07 to 5.59%IFNγ-producing CD8⁺ T cells). In 6 of 17 participants, thesepre-existing responses were slightly amplified upon BNT162b2 dosing.

Overall, the mean fractions of S-specific CD4⁺ and CD8⁺ T cells weresubstantially higher (e.g., the S protein pool 1 IFNγ CD8⁺ response of30 μg dosed participants was 12.5-fold higher) than that observed in 18patients who recovered from COVID-19. Importantly, for the clinicallytargeted 30 μg dose group, the cytokine responses elicited aftervaccination with BNT162b2 in older participants was mostly identical inresponse pattern and intensity with that of the younger participants.

BNT162b2-induced T-cell responses, especially for CD8⁺ T cells, were notlimited to the RBD, and pronounced and strong T cell recognition ofnon-RBD regions of the S protein were observed.

BNT162b2 induced poly-functional and pro-inflammatory CD4⁺/CD8⁺ T-cellresponses in almost all participants. The Th1 polarization of the helperresponse was characterized by a robust IFNγ/IL-2 and only minor IL-4production upon antigen-specific (wild-type SARS-CoV-2 S protein peptidepools) re-stimulation.

Example 25: Certain T Cell Responses Induced by BNT162b2

In addition to Examples 23 and 24, which describe certain T cellresponses induced by immunization with BNT162b2 as observed in theGerman trial (Study BNT162-01; NCT04380701), the present Example furtherdemonstrates immunogenicity of prime-boost vaccination with 1, 10, 20and 30 μg BNT162b2 in participants 19-55 years of age, includingdetailed characterisation of T cell responses, e.g., the firstidentification of epitopes recognised by CD8+ T cells induced by aCOVID-19 vaccine described herein. Without wishing to be bound by anyparticular theory, it is noted that identity of epitopes to which aresponse is raised in a subject, and/or extent of response to particularepitope or combination of epitopes may impact one or more features(e.g., effectiveness and/or duration) of an immune response and/or ofimmune protection provided by an administered vaccine. In someembodiments, an administration regimen may involve one or more steps ofmonitoring one or more features of an immune response, including, forexample, presence and/or level of response (e.g., of T cells and/orantibodies) that recognize one or more particular epitopes. In someembodiments, need for, timing of, and/or magnitude of a subsequent dosemay be determined in light of such monitoring.

As further described below, the present Example demonstrates, in part,that the epitopes recognised by several BNT162b2-induced CD8⁺ T cellswhen presented on frequent MHC alleles were identified using peptide MHCmultimers; and that CD8⁺ T cells were shown to be of theearly-differentiated effector-memory phenotype, with singlespecificities reaching 0.01-3% of circulating CD8⁺ T cells. Withoutwishing to be bound by any particular theory, it is noted that cellsthat exhibit “effector-memory” phenotype may provide longer termprotection.

The present Example also documents that certain participants receivingBNT162b2 had pre-existing T cell responses. Thus, among other things,this example confirms that compositions as described herein, andparticularly BNT162b2 may well be useful even in subjects who havealready been exposed to one or more related viruses, includingpotentially the same virus—i.e., SARS-CoV-2 and/or to an antigen thereofor another antigen that shares one or more epitopes with SARS-CoV2 spikeprotein.

Prevalence and Magnitude of Vaccine-Induced T Cell Responses

T cell responses of 37 BNT162b2 immunised participants from whomsufficient peripheral blood mononuclear cells (PBMCs) were availablewere analysed pre-vaccination (day 1) and seven days after the boosterdose (day 29) by direct ex vivo IFNγ enzyme-linked immunosorbent spot(ELISpot) assay (FIG. 112 and FIG. 113 ). One of ordinary skill in theart will understand that SARS-CoV-2 S protein is composed of a signalpeptide (aa 1-13), the N-terminal S1 protease fragment (aa 14-685), andthe C-terminal S2 protease fragment (aa 686-1273); and that S1 containsthe RBD (aa 319-541), which binds to the host receptor, and that S2mediates fusion between the viral envelope and cell membrane. Todeconvolute reactivity against S protein, CD4⁺ or CD8⁺ T cell effectorswere stimulated overnight with overlapping peptides representingdifferent portions of the wild-type sequence of SARS-CoV-2 S, namelyN-terminal pools ‘S pool 1’ (aa 1-643) and ‘RBD’ (aa 1-16 fused to aa327-528 of S), and the C-terminal ‘S pool 2’ (aa 633-1273).

Seven days after the boost with BNT162b2 at any of the indicated doses,robustly expanded SARS-CoV-2 S-specific CD4⁺ T-cells were detectable inall 37 participants (FIG. 112 a , FIG. 113 a ). In 34 of theseparticipants, comparison to pre-vaccination PBMCs was possible. Thirtyof the 34 subjects (88.2%) had de novo (not existent at baseline) CD4⁺ Tcell responses against both S pool 1 and S pool 2 of SARS-CoV-2. Oneparticipant had de novo response only against pool 2. The remainingthree participants had de novo responses against S pool 1 and lownumbers of pre-existing S pool 2-reactive CD4⁺ T cells. In two of thesethree participants, the pre-existing responses against S pool 2 wereamplified by vaccination (from 91 and 188 spots/10⁶ cellspre-vaccination to 1391 and 965 spots after vaccination, respectively),whereas in one of the three participants, the pre-existing responsesagainst S pool 2 remained stable (53 to 140 spots/10⁶ cells). These datademonstrate that in 94.1% (32/34) of participants, two doses of BNT162b2induce poly-epitopic CD4⁺ T cell responses (de novo or amplified)directed against both N- and C-terminal portions of S and thus againstepitopes outside the RBD (FIG. 113 b ).

Although for dose levels ≥10 μg the magnitude of CD4⁺ T cell responsesdid not appear to be dose-dependent, it varied between individuals. Inthe strongest responders, the S-specific CD4⁺ T cell responses were morethan 10-fold of the individual memory responses to common viruses andrecall antigens (those from cytomegalovirus, Epstein Barr virus,influenza virus and tetanus toxoid) (FIG. 112 b,c ).

Vaccine-induced S-specific CD8⁺ T cell responses were detected in 34 of37 vaccinated participants (91.9%). The majority were strong responses(FIG. 112 a , FIG. 113 a ) comparable to individual memory responsesagainst cytomegalovirus (CMV), Epstein Barr virus (EBV) and influenzavirus (FIG. 112 b,c ). De novo S-specific CD8⁺ T cell responses wereinduced in 33 participants, these were either directed against both (22participants), or one of the S pools (S pool 1 in ten participants, andS pool 2 in two participants), indicating a preponderance of apoly-epitopic response including non-RBD S-specific T cells (FIG. 113 b). In seven participants, pre-existing CD8⁺ T cell responses to S pool 2were detected that were not further augmented by vaccination. Six out ofthese seven participants had a concurrent de novo response to pool 1 ofS, which in strength did not differ significantly from those observed inindividuals without pre-existing responses to S pool 2 (FIG. 113 c ). Ofnote, the strongest responses (higher than third quartile) against Spool 1 among the 34 participants with detectable CD8⁺ T cell responseswere observed in those without pre-existing S pool 2-specific responses.

The magnitude of S-specific CD4⁺ T cell responses correlated positivelywith S1-binding IgG (FIG. 114 a ), and, in line with the concept ofintramolecular help (e.g., a CD4 response to one eptiope in an antigencan support development of a CD8 response to an epitope in the sameantigen), also with the strength of S-specific CD8⁺ T cell responses(FIG. 114 b ). S-specific CD8⁺ T cell responses also correlatedpositively with S1-binding IgG (FIG. 114 c ), indicating a convergentdevelopment of the humoral and cellular adaptive immunity.

Polarisation of Vaccine-Induced T Cell Responses

To assess functionality and polarisation of S-specific T cells,cytokines secreted in response to stimulation with S pool 1, S pool 2and RBD pool were determined by intracellular staining (ICS) for IFNγ,IL-2 and IL-4 specific responses in pre- and post-vaccination PBMCs of37 BNT162b2-immunised participants receiving different doses. Aconsiderable fraction of vaccine-induced, S-specific CD4⁺ T cellssecreted IFNγ, IL-2, or both, while T cells secreting the T_(H)2cytokine IL-4 were barely detectable (FIG. 115 a-c , FIG. 113 d-e ).Vaccine-induced S-specific CD8⁺ T cells secreted predominantly IFNγ andlower levels of IL-2 in response to S pool 1 and S pool 2 stimulation.Fractions of IFNγ⁺ CD8⁺ T cells specific to S pool 1 constituted up toabout 1% of total peripheral blood CD8⁺ T cells (FIG. 115 d ). Of note,several of the analysed participants (n=3 in the 20 μg dose cohort andn=3 in the 30 μg dose cohort) displayed pre-existing S pool 2 specificCD8⁺ T cell responses, which in 5 out of the 6 participants were notfurther amplified after vaccination. A strong pre-existing S pool 2specific IFNγ⁺ CD4⁺ T cell response was detectable in one participant(20 μg dose cohort) (FIG. 115 c ).

In both assay systems, cytokine production of CD4⁺ as well as CD8⁺ Tcells in response to peptide pools comprising the full SARS-CoV-2 Sexceeded the responses against the RBD peptide pool, further confirmingthe poly-epitopic nature of T cell responses elicited by BNT162b2. Themean fraction of BNT162b2-induced S-specific IFNγ⁺ or IL-2⁺ CD4⁺ andCD8⁺ T cells within total circulating T cells was higher than thatdetected in eighteen control subjects who had recovered from COVID-19(HCS) (FIG. 115 c,d ).

Epitope Specificity and Phenotype of CD8⁺ T Cells Observed inImmunization with BNT162b2

CD8⁺ T cell responses were characterised on the epitope level in threeparticipants vaccinated with a 2-dose regimen with two doses (e.g., 10μg/dose or 30 μg/dose) given 21 days apart.

Pre- and post-vaccination peripheral blood mononuclear cells (PBMCs)collected from the participants were stained with individualisedpeptide/MHC multimer staining cocktails for flow cytometry analysis.Twenty-three (4 for HLA-B*0702, 19 for HLA-A*2402), 14 (HLA-B*3501) andtwenty-three (7 for HLA-B*4401, 16 for HLA-A*0201) diverse peptide/MHCallele pairs were used for participant 1, 2 and 3, respectively, thusprobing a selected set of potential reactivities rather thancomprehensively capturing the poly-epitopic T cell response. For eachparticipant, de novo induced CD8⁺ T cell reactivities against multipleepitopes were identified adding up to a total of eight differentepitope/MHC pairs spread across the full length of S protein (FIG. 116a, c). The magnitude of epitope-specific T cell responses ranged between0.01-3.09% of peripheral CD8⁺ T cells and the most profound expansionwas observed for HLA-A*0201 YLQPRTFLL (SEQ ID NO: 40) (3.09% multimer⁺of CD8⁺), HLA-A*2402 QYIKWPWYI (SEQ ID NO: 42) (1.27% multimer⁺ of CD8⁺)and HLA-B*3501 QPTESIVRF (SEQ ID NO: 45) (0.17% multimer⁺ of CD8⁺).Comparison with the bulk IFNγ⁺ CD8⁺ T cell response against full Sprotein in these individuals determined by ELISpot and intracellularstaining (ICS) indicated that pMHC technology may be more useful toassess the true extent of the cellular immune response (FIG. 113 f ).

Phenotyping of the identified pMHC multimer⁺ S antigen-experienced CD8⁺T cell specificities revealed an early differentiated effector memoryphenotype characterized by low expression of CCR7 and CD45RA and highexpression of the costimulatory molecules CD28 and CD27. CD8⁺ T cellsalso expressed markers associated with cognate activation, such as CD38,HLA-DR and PD-1 (FIG. 116 b ).

Discussion

Effectors of the adaptive immune system have complementary roles in thedefense of viral infections. While neutralising antibodies are the firstline of defense, CD8+ cytotoxic T lymphocytes (CTLs) contribute to virusclearance from intracellular compartments that are inaccessible toneutralising antibodies. Antigen-specific CD4+ T cells have immuneorchestrating functions, including provision of cognate help to B cellsand CD8+ T cells, support of memory generation, as well as indirect(e.g. via IFNγ) or direct (against MHC class II-expressing target cells)cytotoxic activity.

This Example shows that vaccination with BNT162b2 induces a coordinatedimmune response with SARS-CoV-2 S-specific neutralising antibodies (asdescribed in other Examples), CD4+ T cells, CD8+ T cells, andimmune-modulatory cytokines such as IFNγ. All participants vaccinatedwith BNT162b2 mounted de novo S-specific CD4+ T cell responses andalmost 92% of participants mounted CD8+ T cell responses, as detectedwith an ex vivo ELISpot assay. The magnitude of the T cell responsesvaried inter-individually and showed no clear dose dependency. Even withthe lowest dose of 1 μg BNT162b2, most of the vaccinated participantsdemonstrated robust expansion of CD4+ and CD8+ T cells. T cell responseswere directed against RBD, S1 and S2 regions of S protein, indicatingimmune recognition of multiple independent MHC I and II epitopes.

Expression of IFNγ and IL-2 but only low levels of IL-4 inBNT162b2-induced CD4+ T cells indicated a TH1 profile and the absence ofa potentially deleterious TH2 immune response.

While all CD8+ T cell responses against the S1 subunit of S protein werede novo and not detected at baseline, pre-existing immune responsesagainst the S2 subunit were identified in several individuals. The S1fragment has less sequence similarity to the corresponding seasonalcoronavirus sequences than the S2 fragment does; without wishing to bebound by theory, it is proposed that this finding indicates thatpre-existing cross-reactive CD8+ T cells may have been detected.

Peptide MHC (pMHC) multimer technology enabled the identification of Sprotein epitopes recognised by vaccine-induced CD8⁺ T cells as well asdirect quantification of the respective epitope-specific T cells. Thecumulative T cell frequencies in each participant exceeded the overall Tcell response measured in ELISpot and ICS assays, indicating that thoseassays underestimate the true magnitude of the poly-epitopic response.One of skill in the art will appreciate that single peptide analyses areknown to yield higher T cell frequencies as compared to functional Tcell assays that stimulate with peptide pools, with a multitude ofimmunogenic epitopes competing. A high proportion of induced CD8⁺ Tcells were early differentiated effector memory cells. This favourablephenotype has the potential to respond rapidly, but has a limitedcapacity to produce IFNγ, and thus is less likely to be detected infunctional T cell assays. While epitopes in SARS-CoV-2 S against whichinfected individuals raise CD8+ T cells were identified and known in theart (see, e.g., Shomuradova et al., Immunity (2020)doi:10.1016/j.immuni.2020.11.004; and Peng et al. Nat. Immunol. 21,1336-1345 (2020)), the data presented herein is the first demonstrationof epitopes recognised by COVID-19 vaccine-induced T cells. Of note, theimmunodominant HLA-A*02:01 restricted peptide YLQPRTFLL (SEQ ID NO: 40)identified in this study has previously been described in convalescentCOVID-19 patients. Id.

Materials and Methods Proteins and Peptides.

Two pools of 15-mer peptides overlapping by 11 amino acids (aa) andtogether covering the whole sequence of wild-type SARS-CoV-2 S (S pool 1featuring aa 1-643, S pool 2 featuring aa 633-1273) and one poolcovering the SARS-CoV-2 RBD (aa 327-528) with the signal peptide of S(aa 1-16) fused to its N-terminus were used for ex vivo stimulation ofPBMCs for flow cytometry and IFNγ ELISpot. CEF (CMV, EBV, influenzavirus; human leukocyte antigen [HLA] class I epitope peptide pool) andCEFT (CMV, EBV, influenza virus, tetanus toxoid; HLA class II epitopepeptide pool) were used as controls for general T-cell reactivity and tobenchmark the magnitude of memory T cell responses. All peptides wereobtained from JPT Peptide Technologies.

Human Convalescent Serum and PBMC Panel.

Human SARS-CoV-2 infection/COVID-19 convalescent sera (n=38) were drawnfrom donors 18-83 years of age at least 14 days after PCR-confirmeddiagnosis and at a time when the participants were asymptomatic. Themean age of the donors was 45 years. Neutralising GMTs in subgroups ofthe donors were as follows: symptomatic infections, 90 (n=35);asymptomatic infections, 156 (n=3); hospitalized, 618 (n=1). Sera wereobtained from Sanguine Biosciences (Sherman Oaks, Calif.), the MT Group(Van Nuys, Calif.) and Pfizer Occupational Health and Wellness (PearlRiver, N.Y.). Human SARS-CoV-2 infection/COVID-19 convalescent PBMCsamples (n=18) were collected from donors 22-79 years of age 30-62 daysafter PCR-confirmed diagnosis, when donors were asymptomatic. PBMCdonors had asymptomatic or mild infections (n=16, clinical score 1 and2) or had been hospitalized (n=2, clinical score 4 and 5). Blood sampleswere obtained from the Frankfurt University Hospital.

Primary Cell Isolation.

PBMCs were isolated by Ficoll-Paque™ PLUS (Cytiva) density gradientcentrifugation and cryopreserved prior to analysis.

IFNγ ELISpot.

IFNγ ELISpot analysis was performed ex vivo (without further in vitroculturing for expansion) using PBMCs depleted of CD4⁺ and enriched forCD8⁺ T cells (CD8⁺ effectors) or depleted of CD8⁺ and enriched for CD4⁺T cells (CD4⁺ effectors). Tests were performed in duplicate and with apositive control (anti-CD3 monoclonal antibody CD3-2 [1:1,000;Mabtech]). Multiscreen filter plates (Merck Millipore) pre-coated withIFNγ-specific antibodies (ELISpotPro kit, Mabtech) were washed with PBSand blocked with X-VIVO 15 medium (Lonza) containing 2% human serumalbumin (CSL-Behring) for 1-5 hours. Per well, 3.3×10⁵ effector cellswere stimulated for 16-20 hours with three overlapping peptide poolsrepresenting different portions of the wild-type sequence of SARS-CoV-2S (N-terminal pools S pool 1 [aa 1-643] and RBD [aa1-16 fused to aa327-528], and the C-terminal S pool 2 [aa 633-1273]). Bound IFNγ wasvisualised using a secondary antibody directly conjugated with alkalinephosphatase followed by incubation with 5-bromo-4-chloro-3′-indolylphosphate (BCIP)/nitro blue tetrazolium (NBT) substrate (ELISpotPro kit,Mabtech). Plates were scanned using an AID Classic Robot ELISPOT Readerand analysed by AID ELISPOT 7.0 software (AID Autoimmun Diagnostika).Spot counts were displayed as mean values of each duplicate. T-cellresponses stimulated by peptides were compared to effectors incubatedwith medium only as a negative control using an in-house ELISpot dataanalysis tool (EDA), based on two statistical tests (distribution-freeresampling), to provide sensitivity while maintaining control over falsepositives.

To account for varying sample quality reflected in the number of spotsin response to anti-CD3 antibody stimulation, a normalisation method wasapplied, enabling direct comparison of spot counts and strength ofresponse between individuals. This dependency was modelled in alog-linear fashion with a Bayesian model including a noise component(unpublished). For a robust normalization, each normalisation wassampled 1000 times from the model and the median taken as normalizedspot count value. Likelihood of the model: log λ_(E)=α log λ_(P)+logβ_(j)+σε, where λ_(E) is the normalized spot count of the sample; α is astable factor (normally distributed) common among all positive controlsλ_(P); β_(j) is a sample j specific component (normally distributed);and σε is the noise component, of which σ is Cauchy distributed, and εis Student's-t distributed. β_(j) ensures that each sample is treated asa different batch.

Flow Cytometry.

Cytokine-producing T cells were identified by intracellular cytokinestaining. PBMCs thawed and rested for 4 hours in OpTmizer mediumsupplemented with 2 μg/mL DNase I (Roche), were restimulated withdifferent portions of the wild-type sequence of SARS-CoV-2 S in peptidepools described in the ELISpot section (2 μg/mL/peptide; JPT PeptideTechnologies) in the presence of GolgiPlug (BD) for 18 hours at 37° C.Controls were treated with DMSO-containing medium. Cells were stainedfor viability and surface markers (CD3 BV421, 1:250; CD4 BV480, 1:50;CD8 BB515, 1:100; all BD Biosciences) in flow buffer (DPBS [Gibco]supplemented with 2% FBS [Biochrom], 2 mM ethylenediaminetetraaceticacid [EDTA; Sigma-Aldrich]) for 20 minutes at 4° C. Afterwards, sampleswere fixed and permeabilised using the Cytofix/Cytoperm kit according tomanufacturer's instructions (BD Biosciences). Intracellular staining(CD3 BV421, 1:250; CD4 BV480, 1:50; CD8 BB515, 1:100; IFNγ PE-Cy7, 1:50[for HCS]; IFNγ BB700, 1:250 [for participants]; IL-2 PE, 1:10; IL-4APC, 1:500; all BD Biosciences) was performed in Perm/Wash buffer for 30minutes at 4° C. Samples were acquired on a fluorescence-activated cellsorter (FACS) VERSE instrument (BD Biosciences) and analysed with FlowJosoftware version 10.6.2 (FlowJo LLC, BD Biosciences). S- andRBD-specific cytokine production was corrected for background bysubtraction of values obtained with dimethyl sulfoxide (DMSO)-containingmedium. Negative values were set to zero. Cytokine production in FIG.116 b was calculated by summing up the fractions of all CD4⁺ T cellspositive for either IFNγ, IL-2 or IL-4, setting this sum to 100% andcalculating the fraction of each specific cytokine-producing subsetthereof. Pseudocolor plot axes are in log 10 scale.

Peptide/MHC Multimer Staining.

In order to select MHC-class I epitopes for multimer analysis, a massspectrometry-based binding and presentation predictor (e.g., asdescribed in Abelin et al., Immunity 46, 315-326 (2017); and Poran etal., Genome Med. 12, 70 (2020)) was applied to 8-12 amino acid longpeptide sequences from the Spike glycoprotein derived from the GenBankreference sequence for SARS-CoV-2 (accession: NC_045512.2,www.ncbi.nlm.nih.gov/nuccore/NC_045512) and paired with 18 MHC-class-Ialleles with >5% frequency in the European population. Top predictedepitopes were identified by setting thresholds to the bindingpercent-rank (51%) and presentation scores (≥10^(−2.2)) and consideredfor synthesis of peptides of >90% purity. pMHC complexes were refoldedwith the easYmer technology (easYmer® kit, ImmuneAware Aps), and complexformation was validated in a bead-based flow cytometry assay accordingto the manufacturer's instructions. Combinatorial labeling was used fordissecting the antigen specificity of T cells utilizing two-colorcombinations of five different fluorescent labels to enable detection ofup to ten different T cell populations per sample. For tetramerisation,streptavidin (SA)-fluorochrome conjugates were added: SA BV421, SABV711, SA PE, SA PE-Cy7, SA APC (all BD Biosciences). For three BNT162b2vaccinated participants, individualized pMHC multimer staining cocktailscontained up to ten pMHC complexes, with each pMHC complex encoded by aunique two-color combination. PBMCs (2×10⁶) were stained ex vivo for 20minutes at room temperature with each pMHC multimer cocktail at a finalconcentration of 4 nM in Brilliant Staining Buffer Plus (BSB Plus [BDHorizon™]). Surface and viability staining was carried out in flowbuffer (DPBS [Gibco] with 2% FBS [Biochrom], 2 mM EDTA [Sigma-Aldrich])supplemented with BSB Plus for 30 minutes at 4° C. (CD3 BUV395, 1:50;CD45RA BUV563, 1:200; CD27 BUV737, 1:200; CD8 BV480, 1:200; CD279 BV650,1:20; CD197 BV786, 1:15; CD4 BB515, 1:50; CD28 BB700, 1:100; CD38PE-CF594, 1:600; HLA-DR APC-R700, 1:150; all BD Biosciences; DUMPchannel: CD14 APC-eFluor780, 1:100; CD16 APC-eFluor780, 1:100; CD19APC-eFluor780, 1:100; fixable viability dye eFluor780, 1:1,667; allThermoFisher Scientific). Cells were fixed for 15 minutes at 4° C. in 1×Stabilization Fixative (BD), acquired on a FACSymphony™ A3 flowcytometer (BD Biosciences) and analysed with FlowJo software version10.6.2 (FlowJo LLC, BD Biosciences). CD8⁺ T cell reactivities wereconsidered positive, when a clustered population was observed that waslabelled with only two pMHC multimer colors.

Example 26: Evidence Suggesting Possibility of Re-Infection

The primary endpoint was evaluated in individuals without prior evidenceof COVID-19 disease, and very few cases of confirmed COVID-19 occurredamong participants with evidence of infection prior to vaccination(although more cases occurred in the placebo group compared with thevaccine group). However, available data, while limited, as shown inTables 22-23 suggest that previously infected individuals can be at riskof COVID-19 (i.e., reinfection) and could benefit from vaccination.

TABLE 22 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days AfterDose 2, by Test Status - Subjects With or Without Evidence of InfectionPrior to 7 Days After Dose 2 - Evaluable Efficacy (7 Days) PopulationVaccine Group Control Group N^(a) = 19965 N^(a) = 20172 Cases Casesn1^(b) n1^(b) Vaccine RT-PCR NP Swab Results and surveillancesurveillance Efficacy % Serostatus: Time Points time^(c) (n2^(d))time^(c) (n2^(d)) (95% CI^(e)) Pre-dose 1 SARS-CoV-2 RT-PCR (NP swab)Positive 0 0 NE (NE, 0.013 (119) 0.015 (137) NE) Negative 9 166  94.5(89.4,  2.301 (18259)  2.314 (18410) 97.6) Unknown 0 3 100.0 (−126.2,0.017 (181) 0.016 (161) 100.0) Pre-dose 2 SARS-CoV-2 RT-PCR (NP swab)Positive 0 1 100 (−4916.4, 0.009 (83)  0.012 (106) 100.0) Negative 9167  94.6 (89.5,  2.301 (18263)  2.315 (18408) 97.6) Unknown 0 1 100.0(−3173.8, 0.022 (213) 0.018 (194) 100.0) Subjects with negative RT-PCRpre-dose 1 and positive RT-PCR pre-dose 2 Subjects with documented 0 0 —COVID-19 symptoms 0.000 (0)  0.000 (1)  between dose 1 and 2 Subjectswith no documented 0 1 — COVID-19 symptoms 0.004 (44)  0.006 (52) between dose 1 and 2 Pre-dose 1 serostatus^(f) Positive 1 1 −8.0(−8378.1, 0.052 (488) 0.056 (525) 98.6) Negative 8 167  95.2 (90.3, 2.255 (17823)  2.260 (17894) 98.0) Unknown 0 1 100.0 (−4527.0, 0.025(248) 0.030 (289) 100.0) 1-month post-dose 2 (Visit 3) No data shownserostatus* Positive Negative Subjects who seroconverted** No data shownbetween dose 1 and 1-month post-dose 2 Subjects with documented COVID-19symptoms during time period Subjects with no documented COVID-19symptoms during time period (protocol- defined vaccine efficacy againstasymptomatic infection) Abbreviations: N-binding = SARS-CoV-2nucleoprotein-binding; RT-PCR = reverse transcription-polymerase chainreaction; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2;VE = vaccine efficacy; NP = nasopharyngeal; NE = Not estimable. ^(a)N =number of subjects in the specified group. ^(b)n1 = Number of subjectsmeeting the endpoint definition. ^(c)Total surveillance time in 1000person-years for the given endpoint across all subjects within eachgroup at risk for the endpoint. Time period for COVID-19 case accrual isfrom 7 days after Dose 2 to the end of the surveillance period. ^(d)n2 =Number of subjects at risk for the endpoint. ^(e)Confidence interval(CI) for VE is derived based on the Clopper and Pearson method adjustedfor surveillance time. ^(f)Based on N-binding test result.

TABLE 23 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days AfterDose 2, by Test Status - Subjects With or Without Evidence of InfectionPrior to 7 Days After Dose 2 - All Available Efficacy Population VaccineGroup Control Group N^(a) = 20488 N^(a) = 20459 Cases Cases n1^(b)n1^(b) Vaccine RT-PCR NP Swab Results and surveillance surveillanceEfficacy % Serostatus: Time Points time^(c) (n2^(d)) time^(c) (n2^(d))(95% CI^(e)) Pre-dose 1 SARS-CoV-2 RT-PCR (NP swab) Positive 0 0 NE (NE,0.014 (122) 0.015 (138) NE) Negative 9 169  94.7 (89.7,  2.358 (18740) 2.338 (18668) 97.6) Unknown 0 3 100.0 (−125.3, 0.018 (187) 0.016 (165)100.0) Pre-dose 2 SARS-CoV-2 RT-PCR (NP swab) Positive 0 1 100.0(−4739.5, 0.010 (86)  0.012 (107) 100.0) Negative 9 170  94.7 (89.8, 2.357 (18743)  2.339 (18660) 97.6) Unknown 0 1 100.0 (−3290.7, 0.022(220) 0.019 (204) 100.0) Subjects with negative RT-PCR pre-dose 1 andpositive RT-PCR pre-dose 2 Subjects with documented 0 0 — COVID-19symptoms 0.000 (0)  0.000 (1)  between dose 1 and 2 Subjects with nodocumented 0 1 — COVID-19 symptoms 0.005 (47)  0.006 (53)  between dose1 and 2 Pre-dose 1 serostatus^(f) Positive 1 1 −7.5 (−8335.1, 0.053(500) 0.057 (537) 98.6) Negative 8 170  95.3 (90.6,  2.308 (18278) 2.283 (18144) 98.0) Unknown 0 1 100.0 (−4081.4, 0.028 (271) 0.030 (290)100.0) 1-month post-dose 2 (Visit 3) No data shown serostatus* PositiveNegative Subjects who seroconverted** No data shown between dose 1 and1-month post-dose 2 Subjects with documented COVID-19 symptoms duringtime period Subjects with no documented COVID-19 symptoms during timeperiod (protocol- defined vaccine efficacy against asymptomaticinfection) Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding;RT-PCR = reverse transcription-polymerase chain reaction; SARS-CoV-2 =severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy;NP = nasopharyngeal; NE = Not estimable. ^(a)N = number of subjects inthe specified group. ^(b)n1 = Number of subjects meeting the endpointdefinition. ^(c)Total surveillance time in 1000 person-years for thegiven endpoint across all subjects within each group at risk for theendpoint. Time period for COVID-19 case accrual is from 7 days afterDose 2 to the end of the surveillance period. ^(d)n2 = Number ofsubjects at risk for the endpoint. ^(e)Confidence interval (CI) for VEis derived based on the Clopper and Pearson method adjusted forsurveillance time. ^(f)Based on N-binding test result.

Example 27: Pharmacokinetics (PK) and Absorption, Distribution,Metabolism, and Excretion (ADME) Analysis of Certain Lipid Excipients

The present Example describes various assessed characteristics (e.g.,PK/ADME characteristics) of lipids used in a vaccine composition asdescribed herein. Without wishing to be bound by any particular theoryit is noted that such characteristics of lipid components may contributeto relevant features (e.g., distribution, expression, etc) ofadministered vaccines, including to efficacy generally and/or inparticular circumstances (e.g., when administered according toparticular regimens and/or to particular populations, etc).

Absorption

A single dose PK study of ALC-0315 and ALC-0319 following intravenous(IV) bolus injection of a nanoparticle formulation in rats was conductedto assess the PK and metabolism of lipid excipients ALC-0315 andALC-0159. This study used LNPs containing surrogate luciferase RNA, withthe lipid composition being identical to BNT162b2, to investigate the invivo disposition of ALC-0159 and ALC-0315.

Concentrations of ALC-0159 dropped approximately 8000- and >250-fold inplasma and liver, respectively, during this 2-week study. For ALC-0315,the elimination of the molecule from plasma and liver was slower, butconcentrations fell approximately 7000- and 4-fold in two weeks forplasma and liver, respectively. Overall, the apparent terminal t½ inplasma and liver were similar in both tissues and were 2-3 and 6-8 daysfor ALC-0159 and ALC-0315, respectively. The apparent terminal t½ inplasma likely represents the re-distribution of the respective lipidsfrom the tissues into which they have distributed as the LNP, back toplasma where they are eliminated.

Metabolism

In vitro metabolism of ALC-0315 and ALC-0159 was evaluated in blood,liver microsomes, S9 fractions, and hepatocytes from mice, rats,monkeys, and humans. In vivo metabolism was examined in rat plasma,urine, faeces, and liver samples from the PK study. Metabolism ofALC-0315 and ALC-0159 appears to occur relatively slowly in vitro and invivo. ALC-0315 and ALC-0159 are apparently metabolised by hydrolyticmetabolism of the ester and amide functionalities, respectively, andthis hydrolytic metabolism is observed across the species evaluated.

Excretion

Excretion studies appeared to demonstrate that 50% of ALC-0159 waseliminated unchanged in faeces, and that metabolism played a role in theelimination of ALC-0315, as little to no unchanged material was detectedin either urine or faeces. Investigations of urine, faeces and plasmafrom the rat PK study identified a series of ester cleavage products ofALC-0315. Without wishing to be bound by any particular theory, it isproposed that this likely represents the primary clearance mechanismacting on this molecule in vivo. In vitro, ALC-0159 was metabolizedslowly by hydrolytic metabolism of the amide functionality.

Example 28: Distribution Analysis of Administered Vaccine Composition

In vivo biodistribution of COVID-19 mRNA Vaccine BNT162b2 was evaluatedusing mice as a model system and assessing luciferase expression as asurrogate reporter. Protein expression was demonstrated at the site ofinjection and to a lesser extent, and more transiently, in the liverafter mice received an IM injection of RNA encoding luciferase in an LNPformulation like BNT162b2. Luciferase expression was identified at theinjection site at 6 hours after injection and diminished to nearbaseline levels by day 9. Expression in the liver was also present at 6hours after injection and was not detected by 48 hours after injection.All other tissues than liver evaluated contain equal to or less than 1%of the dose.

Example 29: Repeat-Dose Toxicity Study of Various Dosing Regimens

A GLP-compliant repeat-dose study performed in rats to evaluateimmunogenicity and toxicity of COVID-19 mRNA vaccines, includingBNT162b2.

In certain studies, male and female Wistar Han rats were given a vaccinecomposition as described herein; compositions based on various RNAplatforms (e.g., BNT162b2) were tested as IM injection(s) into the hindlimb on three occasions each a week apart (dosing days 1, 8 and 15).Different doses (10, 30 and 100 μg) were tested; the lower doses weregiven as a single injection of 20-70 μl while the highest doses (100 μg)and controls were given as two injections (one in each hind limb) of 100μl each. The control was phosphate buffered saline/300 mM sucrose,corresponding to the storage buffer of the vaccine product. Each grouphad 18 male and 18 female rats, assigned as 10 to the main study, 5 forrecovery groups and 3 as additional animals for cytokine analyses. Therecovery period was 3 weeks after the last dose. Necropsy was performedon study day 17, ^(˜)48 hours after the last dose and after the 3-weekrecovery period.

No unscheduled deaths were observed.

Dosing was considered well tolerated and did not present any signs ofsystemic toxicity; there was a slight increase in body temperature inthe hours after dosing and some loss in body weight over the same periodbut these were not of a magnitude to be considered adverse.

Local inflammatory reactions were observed at the intramuscularinjection site. Injection site changes noted were oedema, erythema, andinduration, more severe and more frequent after the second and/or thirddoses compared to the first; however, these resolved prior to subsequentdosing and were fully recovered at the end of the 3-week recoveryperiod.

Macroscopic findings at the injection sites included induration orthickening, occasionally accompanied by encrustation, which was notedfor nearly all rats. This correlated microscopically with inflammationand variable fibrosis, oedema, and myofibre degeneration. Inflammationat the injection site was accompanied by elevations in circulating whiteblood cells and acute phase proteins (fibrinogen, alpha-2 macroglobulin,and alpha-1 acid glycoprotein).

Inflammation was occasionally evident extending into tissues adjacent tothe injection site. There was enlargement of the draining (iliac) lymphnodes evident at the end of dosing. This correlated with increasedcellularity of germinal centres and increased plasma cells in thedraining (iliac) lymph node and is an anticipated immune response to theadministered vaccine.

Enlargement of spleen and increased spleen weights correlatedmicroscopically to increased haematopoiesis and increased haematopoiesiswas also evident in the bone marrow. These findings are likely secondaryto the immune/inflammatory responses to the vaccine.

At the end of the recovery period, injection sites were normal, clinicalpathology findings and macroscopic observations had resolved and therewas evidence of recovery of the injection site inflammation onmicroscopy.

Microscopic vacuolation of portal hepatocytes was present after thedosing phase. This observation was absent after the recovery period.There were no elevations in alanine aminotransferase (ALAT). There wereelevations in gamma-glutamyltransferase (GGT) in all vaccinated rats,but there were no macroscopic or microscopic findings consistent withcholestasis or hepatobiliary injury to explain the increased gamma-GTactivity which was completely resolved at the end of the 3-week recoveryperiod. The vacuolation may be related to hepatic distribution of thepegylated lipids in the LNP. No changes were seen in serum cytokineconcentrations. There were no effects noted on ophthalmological andauditory assessments, nor on external appearance or behaviour; inparticular, gait was normal meaning that the changes seen did not affectthe rats' mobility. No vaccine-related changes were seen in serumcytokine concentrations.

Testing for immunogenicity showed that COVID-19 mRNA Vaccines(including, e.g., BNT162b2 such as BNT162b2 v8) elicited a specific IgGantibody response to SARS CoV-2 spike protein directed against the S1fragment and the receptor binding domain. A neutralizing antibodyresponse was also observed with the vaccine in a pseudovirusneutralization assay.

Results from ELISA assays are shown in FIGS. 117 and 118 (from day 17 orday 10 as noted in the figures), in which the very top traces are thosefor COVID-19 mRNA Vaccine BNT162b2 and other traces are those for otherCOVID-19 mRNA vaccines using different constructs as described herein:similar results were shown for day 38 (not shown here). These translatedinto neutralising activity as seen in the VSV/SARS-CoV2-S pseudovirusneutralisation test using Vero 76 cells (FIG. 119 ): similar resultswere presented for day 38 (not shown here). Across the vaccines testedin this study those with a higher antigen-specific antibody titre alsohad a more pronounced virus neutralisation effect.

COVID-19 mRNA vaccines (e.g., BNT162b2) were well tolerated, andproduced inflammatory changes at the injection sites and the draininglymph nodes, increased haematopoiesis in the bone marrow and spleen, andclinical pathology changes consistent with an immune response orinflammation in the injection sites.

Those skilled in the art, reading the present disclosure, willappreciate that the findings in this Example can be considered typicalof those expected with dosing of various mRNA constructs and/or lipidnanoparticles as described herein.

Example 30: Toxicity and Immunogenicity Study of Three-Dose Regimen

A study was performed to assess toxicity in rats given COVID-19 mRNAVaccine (e.g., BNT162b2). This study was in compliance with GoodLaboratory Practice.

Male and female Wistar Han rats were given BNT162b2 as an IM injectioninto the hind limb on three occasions, each a week apart (dosing days 1,8 and 15). Necropsy was performed on study day 17, ˜48 hours after thelast dose, and after the 3-week recovery period. COVID-19 mRNA VaccineBNT162b2 was supplied at 0.5 mg/ml and the dose volume was 60 μl, togive 30 μg per dose. Control rats received saline.

Blood was taken at various points during the assessment, prior to andduring dosing, and also during recovery, and antibody responses tovaccine components were assessed.

All rats given COVID-19 mRNA Vaccine (e.g., BNT162b2) survived to theirscheduled necropsy: there were no changes noted in clinical signs orbody weight changes noted. A reduction in food intake was noted on days4 and 11 (to 0.83× controls) and there was an increase in mean bodytemperature post-dose on day 1 (up to 0.54° C.), day 8 (up to 0.98° C.),and day 15 (up to 1.03° C.) compared to controls.

At injection sites, there were instances of oedema and erythema on days1 (maximum of slight oedema and very slight erythema), 8 (maximum ofmoderate oedema and very slight erythema) and 15 (maximum of moderateoedema and very slight erythema) which fully resolved and were not notedprior to dosing on days 8 and 15. Haematological tests showed higherwhite blood cells (up to 2.95× controls), primarily involvingneutrophils (up to 6.80× controls), monocytes (up to 3.30× controls),and large unstained cells, LUC, (up to 13.2× controls) and slightlyhigher eosinophils and basophils on days 4 and 17. White blood cellswere higher on day 17 as compared with day 4. There were transientlylower reticulocytes on day 4 (to 0.27× controls) in both sexes andhigher reticulocytes on day 17 (up to 1.31× controls) in females only.Lower red blood cell mass parameters (to 0.90× controls) were present ondays 4 and 17. There were lower A:G ratios (to 0.82×) on days 4 and 17.Higher fibrinogen was noted on day 17 (up to 2.49×) compared tocontrols, consistent with an acute phase response. The acute phaseproteins alpha-1-acid glycoprotein (up to 39× on day 17) and alpha-2macroglobulin (up to 71× on Day 17) were elevated on days 4 and 17 withhigher concentrations in males. There were no changes urinalysisparameters.

At post-mortem there were higher absolute and relative spleen weights invaccinated rats (up to 1.42× in males and to 1.62× in females). Therewere no other changes in organ weights. Macroscopic findings includedenlarged draining lymph nodes and pale/dark firm injection sites in aminority of vaccinated rats. The dosing was tolerated without inducingany systemic toxicity with all changes consistent with an inflammatoryresponse and immune activation: findings are consistent with thosetypically associated with dosing of lipid nanoparticle-encapsulated mRNAvaccines.

Example 31: Reproduction Toxicity

A study was performed to assess reproduction toxicity in female ratsgiven COVID-19 mRNA vaccines, including BNT162b2. Female rats were givena COVID-19 mRNA vaccine (e.g., BNT162b2) twice before the start ofmating and twice during gestation at a human clinical dose (e.g., 30 μgRNA/dosing day). The COVID-19 mRNA vaccine was administeredintramuscularly (IM) to F0 female Wistar rats 21 and 14 days before thestart of mating (M-21 and M-14, respectively) and then on Gestation Day(GD) 9 and GD20, for a total of 4 doses. A subgroup was terminated atGD21 and another (litter) group was terminated at PostNatal Day (PND)21. SARS-CoV-2 neutralizing antibody titers were found in the majorityof females just prior to mating (M-14), in most females and foetuses atthe end of gestation (GD21), and in most offspring at the end oflactation (PND21). There was transient reduced body weight gain and foodconsumption after each dose. No effects on the estrous cycle orfertility index were observed. While there was an increase (^(˜)2×) ofpre-implantation loss (as compared to control), the pre-implantationloss percent observed in the vaccinated group was within historicalcontrol data range (5.1%-11.5%). Among foetuses (from a total of n=21dams/litters), there was a very low incidence of gastroschisis,mouth/jaw malformations, right sided aortic arch, and/or cervicalvertebrae abnormalities. Regarding skeletal findings, the exposed grouphad comparable to control group levels of presacral vertebral archessupernumerary lumbar ribs, supernumerary lumbar short ribs, caudalvertebrae number <5). There were no signs of adverse effects on thepostnatal pups (terminated at PND21). This study shows that there is nosignificant adverse effects on fertility and early embryogenesis.

Example 32: Safety and Immunogenicity of the SARS-COV-2 BNT162b1 mRNAVaccine in Younger and Older Chinese Adults: A Randomized,Placebo-Controlled, Observer-Blind Phase I Study

The present Example reports initial results from a phase I trial testingBNT162b1 in 144 healthy Chinese participants. BNT162b1 encodes theSARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) and is oneof several RNA-based SARS-CoV-2 vaccine candidates described herein.

The present Example specifically reports on the observed safety profile,in whichever >39° C. was the only Grade 3 adverse event reported.Prime-boost vaccination with 10 μg or 30 μg BNT162b1 induced robustantibody and T-cell responses in both young (18 to 59 years of age) andolder (65 to 85) Chinese adults. Both dose levels induced seroconversionafter 41 d: Geometric mean titres of SARS-CoV-2 serum-neutralizingantibodies in younger participants in the 10 μg and 30 μg dose groupswere 1.9 and 2.1 times that of convalescent sera from recovered COVID-19patients; and in older participants 0.7 and 1.3 times. Interferon-γ Tcell responses to RBD antigen challenge were significantly higher inparticipants receiving BNT162b1 than those in placebo group.

Increased reactogenicity as well as a more favorable vaccine-elicitedvirus-neutralizing response were associated with the 30 μg dose ofBNT162b1 in both younger and older Chinese adults.

The safety and immunogenicity data provided in the present Example forBNT162b1, specifically with respect to vaccination of healthy, young andelderly Chinese participants, suggests that prime-boost vaccination with10 μg and 30 μg dose levels of the BNT162b1 vaccine induces a stronghumoral and cellular immunogenic response in both younger adults of 18to 55 years of age and older adults of 65 to 85 years of age, withrobust RBD-specific antibody and T-cell responses seen in in bothyounger and older participants, at least within 28 days following theprime-boost vaccination. Certain findings in this study further confirmaspects of the tolerability profile for BNT162b1, for example as alsoobserved in American and German populations.

Methods

A randomized, placebo-controlled, observer-blind phase 1 trial wasconducted in 144 healthy young adults, 18 to 59 years of age, and olderadults 65 to 85 years of age in Taizhou, Jiangsu Province, China.Eligible participants were randomized to receive two doses, administered21 days apart, of either BNT162b1 at a dose of 10 μg or 30 μg orplacebo, administered as an intramuscular injection. Study participants,investigators, and laboratory staff were blinded to treatmentadministration. The primary safety endpoints were local reactions atinjection site or systemic adverse reactions within 14 days aftervaccination, and adverse events occurring up to 28 days after receivingthe boost vaccination. The immunogenic endpoints of virus-neutralizingantibody, and antigen-specific binding antibodies and cellular immuneresponses elicited by vaccine were measured at predefined timepoints.

Results Study Design and Analysis Set

A total of 296 adults aged between 18-55 years or 65-85 years werescreened at Taizhou vaccine clinical research center in JiangsuProvince, in China. 144 eligible participants consented to participatein the trial and were randomized 1:1:1 to receive prime and boost dosesof BNT162b1 at 10 μg or 30 μg, or two placebo doses 21 days apart.Following priming doses, two participants between the ages of 65 and 85years had withdrawn from boost dose administration (one at 10 μg, one at30 μg). The demographic characteristics of the participants are shown inTable 24. The mean age among the younger participants ranged from 37.9to 42.0 years, and the mean age among the older participants ranged from68.5 to 70.7 years in the treatment groups, with equal genderdistribution across treatment groups. The medical history or existingunderlying disorders of the participants were similar across treatmentgroups, with the exception of hypertension, which was noted in olderparticipants at baseline.

Observed Safety and Tolerability Data

Within 14 days after completion of dosing, 21 (88%) of the youngerparticipants in the 10 μg BNT162b1 dose group and 24 (100%) of theyounger participants in the 30 μg BNT162b1 dose group reported at leastone solicited adverse reaction, versus 17% of the younger participantsin the placebo group (Tables 25A-25B). Reactogenicity was dose leveldependent and most evident in the 30 μg BNT162b1 dose group. The mostcommon solicited adverse reactions reported were injection site pain,fever, headache, fatigue, malaise, joint pain, muscle pain chills. Theadverse events were transient and either managed with simple standard ofcare management, or resolved spontaneously. Most of the reported adversereactions were mild or moderate in severity, and resolved within thefirst seven days after each BNT162b1 dose. No injection site reactionswere graded as severe (grade 3). All of the grade 3 systemic adversereactions associated with the vaccination were fever, predominantlyobserved in the younger participants. One male participant in the olderage group experienced an episodic Grade 3 fever accompanied by pain andpruritus at the injection site after administration of the primeBNT162b1 dose at 30 μg, and electively withdrew from boost vaccinationadministration.

No pre-specified trial-halting rules were met during the study. Only oneserious adverse event was reported by a participant of 67 years of age(a humerus fracture caused by a car accident, preventing the participantfrom receiving the boost dose) which was considered as not related tothe vaccine or study procedure. The overall frequencies of injectionsite adverse reactions post-vaccination were comparable after theBNT162b1 prime and boost doses. Some systematic adverse reactions suchas fever, headache, fatigue, and malaise occurred more commonly afterthe BNT162b1 boost dose than after the prime dose in younger adults. Incontrast to the younger participants, elderly participants did notpresent with increased reactogenicity after the BNT162b1 boost dose.

There were no changes reported in blood pressure and respiratory ratesamong the participants across different treatment groups before andafter BNT162b1 administration. Transient increases of temperature andpulse rate 24 hours post-vaccination were noted in both younger andolder participants, especially in the 30 μg dose group. The most commonabnormalities in laboratory values from baseline were transientdecreases in lymphocyte and platelet counts and increases in C-reactionprotein. All laboratory abnormalities were self-limited and resolved ina short period of time without clinical manifestations. These data areconsistent with certain findings reported in other populations (e.g., asdescribed elsewhere herein).

Vaccine-Induced Antibody Responses

All participants were seronegative at baseline (day 1, pre-vaccination),and were monitored for seroconversion at days 8, 22, 29 and 43 byanalyses of SARS-CoV-2 neutralizing antibodies and RBD and S1 proteinbinding antibodies. The BNT162b1 induced antibody responses invaccinated participants were compared with a panel of human COVID-19convalescent serum obtained at least 14 days after PCR-confirmeddiagnosis from 28 COVID-19 patients. The highest neutralization titerswere observed on day 43 (i.e., 21 days after the BNT162b1 boost dose),indicating a continuous uptrend in this group of Asian participantsafter day 29, which seems to further increase in this Asian populationat day 43 among older participants as compared with reports in otherpopulations, showing peak titres occurring earlier and subsequentlysubsiding in this subject population. On day 43, both 10 μg and 30 μgBNT162b1 dose levels induced significant virus-neutralizing antibodyresponses after the BNT162b1 prime dose which was boosted by the secondBNT162b1 dose, with geometric mean titers (GMTs) of 232.9 (95% CI 151.3to 358.5) and 254.0 (184.6 to 349.4) in the younger participants, and80.0 (49.2 to 130.2) and 160.0 (96.7 to 264.6) in the older participantsin the 10 μg and 30 μg dose groups, respectively (FIG. 120 ). Thevirus-neutralizing responses of younger participants in the 10 μg and 30μg dose groups were 1.9 and 2.1 times the GMT of a panel of theconvalescent sera (GMT, 119.9; 95% CI, 70.4 to 203.9). In the olderparticipants, the corresponding ratios were 0.7 and 1.3 times in the 10μg and 30 μg dose groups, respectively. All the younger recipientsshowed positive seroconversion on Day 43, and the seroconversion ratewas 91% at the 10 μg dose and 96% at the 30 μg dose in the olderrecipients on Day 43, respectively. Participants who received the 30 μgdose appeared to have somewhat higher virus-neutralizing antibodyresponses than those received the 10 μg dose. However, the olderparticipants between the ages of 65 and 85 generally showed a slowervirus-neutralizing response and lower peak response than the youngerparticipants between the ages 18 and 55.

Similarly, both doses of BNT162b1 induced high levels of S1- andRBD-binding IgG in the participants after the prime-boost regimen. TheS1- and RBD-binding IgG levels after vaccination across all timepointsin the vaccine recipients were highly correlated with the neutralizingtiters regardless of the age and dose groups, with a correlationcoefficient of 0.85, and 0.79 (p<0.0001), respectively.

Vaccine-Induced T-Cell Responses

Vaccine-induced CD8+ T cell responses in individuals immunized withBNT162b1 were characterized before the priming vaccination (day 1), onday 29 (7 days after the boost vaccination) and on day 43 (21 days afterthe boost vaccination), using a direct ex vivo IFNγ enzyme-linkedimmunosorbent spot (ELISpot) assay with peripheral blood mononuclearcells (PBMCs). At day 29, specific IFN-γ ELISpot responses against theSp1 peptide pool (covering RBD) were significantly higher inparticipants receiving BNT162b1 than those in placebo group (FIG. 121 ).Younger participants aged 18 to 55 years had average spot-forming cellsof 227.5 (95% CI, 146.5 to 308.5) in those who had received 10 μgvaccinations, and 223.5 (181.2 to 265.9) in those who had received the30 μg vaccinations per 105 PBMCs. In older participants aged 65 to 85years, a slightly lower spot-forming cells with averages of 156.5 (84.1to 229.0) and 171.9 (113.4 to 230.3) were noted post-vaccination acrossthe two dose groups. At day 43, younger participants receiving theprime-boost BNT162b1 regimen tended to show a mild decrease in theirS1-specific IFN-γ ELISpot response compared to that seen on day 29; noblood samples were collected at this time point from the olderparticipants, as so this data is not available. No differences betweenthe BNT162b1 and the placebo groups were observed for IFN-γ ELISpotresponses to the Sp2 peptide pool (which does not include peptides ofthe RBD encoded by BNT162b1) and minor non-specific responses to CD8+ Tcells were observed in both dose groups.

Discussion

The trial described in this Example was conducted in China in parallelwith other BNT162 vaccine candidates in multiple regions¹⁴. One focus ofthe study was to establish data with respect to safety andimmunogenicity of mRNA vaccines in Asian populations. This Examplereports a first evaluation of both the safety and immunogenicityprofiles of such an mRNA vaccine in a Chinese population, andfurthermore of younger and older Chinese populations.

This is a preliminary report for the clinical trial of themodified-RNA-based SARS-CoV-2 vaccine candidate BNT162b1, which encodesthe SARS-CoV-2 RBD, administrated to a healthy adult Chinese population.BNT162b1, like BNT162b2 (modRNA encoding the S protein derived from thesame nucleoside-modified platform) induces strong vaccine-inducedantibody responses and strong T cell responses. Clinical safety andimmunogenicity for both BNT162b1 and BNT162b2 candidates have beenevaluated in healthy adults in both German (younger adults; BNT162-01)and American (younger adults and elderly adults aged 65 to 85 years;BNT162-02) populations. In younger adult groups, severe localreactogenicity AEs within 7 days were fewer in American study(BNT162-02) and the present study (BNT162-03) as compared with theGerman study (BNT162-01). Systemic reactogenicity AEs within 7 days werebroadly similar across studies. Systemic AEs (independent ofrelatedness) within 28 days post Dose 2 were slightly higher in theBNT162-02 and BNT162-03 studies, as compared to the BNT162-01 study.

In older adult groups, severe local reactogenicity AEs within 7 dayswere similar across studies. Systemic reactogenicity AEs within 7 dayswere slightly lower in the BNT162-03 study, as compared to the BNT162-01and the BNT162-02 studies. Systemic AEs (independent of relatedness)within 28 days post Dose 2 were slightly higher in the BNT162-03 studyas compared to the BNT162-02 study, however the severe AEs were lower.In summary, comparative analyses of the BNT162b1 safety profile betweenthe BNT162-01, BNT162-02 and BNT162-03 studies at 30 μg showed agenerally comparable profile, and in the systemic reactogenicity/olderpopulation even a better safety profile in the Asian population vsnon-Asian. Thus, findings reported here further complements and expandsreporting of BNT162b1 and other RNA-based vaccine candidates fromclinical trials conducted in Germany and the United States^(7, 8, 15).

The rationale for this study was to evaluate whether intrinsic andextrinsic differences between German and Chinese population have anyimpact on tolerability or immune responses to this novel type ofvaccine. The safety profile of the vaccine candidate BNT162b1 in healthyChinese adults observed in our study appear to be better than thatreported in other populations, in term of severe reactogenicity by localand systemic reactions^(7, 15). Body habitus, endogenous antibodyrepertoire may have an influence. The reactogenicity of BNT162b1 wasdose-dependent. Increased frequencies of adverse events were observedafter administration of the boost vaccination compared with those afterthe prime vaccination, especially in the younger participants. Olderadults had lower incidences of adverse reactions than the youngerparticipants. Grade 3 fever was reported by 17% of the youngerparticipants and 8% of the older participants receiving 30 μg dose,respectively. Nearly all severe fever reactions were transient andself-limiting. One participant had withdrawn from the boost vaccinationdue to the reactions after the prime dose administration, havingepisodic fever or cold intolerance with or without temperature recordaccompanied by the injection site pain, itching and pruritus, whichlasted over two weeks and resolved after taking Hydrocortisone Butyrateointment. Transient decreases in lymphocyte counts as pharmacodynamicmarkers were predominantly observed in the younger recipients at 30 μgdose level of BNT162b1, which was associated with the redistribution oflymphocytes into lymphoid tissues by innate immune stimulation¹⁶.

Both doses of the vaccine candidate BNT162b1 were effective at elicitingspecific humoral and cellular immune responses, with a clear boosteffect of the second vaccination on antibody titers found in bothyounger and older adults. BNT162b1 administered at a 30 μg dosefollowing a prime-boost regimen induced an optimal level of immuneresponses in terms of virus-neutralizing antibody to SARS-CoV-2, whichwas higher than those in a panel of human convalescent serum samples,regardless of age. The humoral response in the Chinese participantsshowed a unique temporal pattern and peaked at day 43 in both agegroups. Although the number of participants was small, andmethodological differences in measurements that may occur can influenceobserved results, the findings reported here suggest that there may be apopulational difference in response to the vaccine.

Since the vaccine candidate BNT162b1 is a modified RNA vaccine encodinga trimeric version of the RBD, the vaccine recipients in the studyreported in the present Example demonstrated significant T-cellresponses specific to S1 peptide pool (containing 166 15-mer S1 peptidesof from the human SARS-CoV-2 virus), but not to the S2 peptide pool. Theresults indicated the cellular responses elicited by BNT162b1 wasantigen specific. By contrast, the vaccine candidate BNT162b2 spectrumwas different from other RNA-based SARS-CoV-2 vaccine, inducing T-cellresponses could recognize both S1 and S2 peptide pools¹⁵. Nevertheless,data showed BNT162b1 at the 30 μg dose was highly immunogenic capable ofeliciting strong humoral and cell-mediated responses in healthy Chineseadults.

Those skilled in the art appreciate that small sample size and agerestriction of 18 years and older may limit the conclusive rigor offindings observed in the present Example. Regardless, given thatprophylactic RNA vaccines as described herein represent a novel approachto vaccination, safety assessments, including in particular populations(e.g., in children and adolescent populations) are particularlyvaluable. Also, although comparison of serum neutralizing responseselicited by the vaccine candidates described herein with that in humanconvalescent serum panels provides meaningful assessment of thevaccines, the level of serological immunity needed to protect againstCOVID-19 has not yet been rigorously established¹⁷. Those skilled in theart also recognize that the human convalescent serum panels that havebeen used in different trials are not standardized among laboratories,and thus may have a different distribution of patient characteristicsand timepoints of collection, so that direct comparison of results(e.g., characterizing different vaccine candidates and/or characterizingvaccine candidates relative to different convalescent serum) may not beinformative.

In summary, results described in the present Example confirmed thedose-dependency safety and good immunogenicity profile of the RNA-basedSARS-CoV-2 vaccine candidate BNT162b1 and further expand the previousfindings for BNT162b1 in the Germany and the United Statestrials^(7, 8, 15). Increased reactogenicity as well as a more favorablevaccine-elicited virus-neutralizing response were found associated withthe 30 μg of the BNT162b1 in both younger and older adults. In contrast,another vaccine candidate BNT162b2 manufactured from same platform,showed a more favorable safety profile⁸. BNT162b1 encodes a relativelysmall RBD immunogen, which might induce a narrower spectrum ofneutralizing antibodies that are less robust to potential antigenicdrift of SARS-CoV-2, compared with BNT162b2, which encodes a full-lengthspike immunogen¹⁸. It is worth noting that the candidate BNT162b2 hasbeen found to be more than 95% effective in preventing COVID-19 inparticipants, with no decreasing efficacy in those over 65 years ofage¹⁹.

Methods Study Design and Participants

This randomized, placebo-controlled, observer-blind phase I trial wasperformed in healthy young adults between 18 and 59 years old, and olderadults between 65 and 85 years of age, in Taizhou, Jiangsu Province,China. Participants were in overall good health established by medicalhistory, physical examination, and laboratory tests at the screeningvisit. Both males and females were included and agreed to usecontraception during the trial. We excluded participants that werepregnant or breastfeeding. Participants that tested positive forSARS-CoV-2 via a commercial rapid diagnostic kit for IgM/IgG antibody toSARS-CoV-2 (manufactured by Livzon diagnostics inc., Zhuhai, China), orvia testing with a pharyngeal swab nucleic acid diagnostic test(manufactured by Fosun pharma, Shanghai, China) were excluded. Imagingfeatures of COVID-19 present in a chest CT scan was a further exclusioncriteria. Participants with serious cardiovascular disease or chronicconditions such as uncontrolled diabetes and hypertension, humanimmunodeficiency virus, hepatitis B and hepatitis C were excluded.Written informed consent was obtained from each participant before thestart of the study.

The study was conducted in accordance with the Declaration of Helsinkiand Good Clinical Practice. The trial protocol was reviewed and approvedby the National Medical Products Administration, China, and theinstitutional review board of the Jiangsu Provincial Center of DiseaseControl and Prevention.

Randomization and Blinding

Eligible participants between 18 and 55 years of age were enrolled inthe younger age group, and older participants aged greater than or equalto 65 years and less than or equal to 85 years were enrolled in theolder age group. Participants were randomized in a ratio of 1:1:1 toreceive the low-dose BNT126b1 or high-dose BNT126b1 or placebo.Participants were stratified by gender, using a Web-based interactiveresponse technology (IRT) system. The blocked randomization list wasgenerated by an independent statistician using SAS software (version9.4).

Authorized unblinded pharmacists prepared the vaccines or placeboaccording to the allocation of participants through the IRT system, andnurses administrated the investigational products to participants. Theunblinded staff had no further involvement in the trial, and wereforbidden to disclosure allocation information to others. All otherinvestigators, participants, laboratory staff and the sponsor remainedblinded throughout the trial.

Vaccine and Vaccination

BNT162b1 as administered consisted of a Good Manufacturing Practice(GMP)-grade mRNA drug substance encoding the trimerized SARS-CoV-2 spikeglycoprotein RBD antigen, formulated with lipids to obtain the RNA-LNPdrug product. Vaccine was transported and supplied as a buffered-liquidsolution for intramuscular injection, and stored at −80° C., e.g., asdescribed herein.

The low-dose and high-dose BNT126b1 contained 10 μg and 30 μg activeingredient, respectively, and the placebo was a commercial salinesolution. Each participant received a prime-boost dosing regimen ofvaccine candidate BNT162b1 at either 10 μg/0.5 ml or at 30 μg/0.5 ml orplacebo of 0.5 ml administered into the deltoid, 21 days apart.

Monitoring of Safety and Immunogenicity

Each participant was asked to remain at the study site for at least sixhours post vaccine administration to donate blood samples prior to and24 hours post prime vaccination and again prior to and 8 days post boostvaccination for laboratory testing. Vital signs including temperature,blood pressure, pulse, and respiratory rate were measured at baseline,and one hour, three hours and six hours post-vaccination. Any adverseevents following the vaccination were documented by participants usingdiaries until day 28 post-administration of the boost dose. Youngergroup participants were enrolled and received the vaccination first.Enrollment of the older age group was launched following evaluation ofthe preliminary safety data of the younger age group for the first 14days post-prime vaccination. Severity of adverse events and laboratoryabnormal changes are graded with both the scale issued by the ChinaState Food and Drug Administration²⁰ and the U.S. Food and DrugAdministration (FDA)²¹. Serum and PBMCs were collected before thevaccination, at day 8 and/or day 22 after the boost dose, to facilitatemeasurement of specific IgG antibody responses to RBD and spikeglycoprotein S1, neutralizing antibody to SARS-CoV-2, and T-cellresponses. All reported adverse events were reviewed by investigators.Adverse events were categorized as either possibly, probably, ordefinitely related to the vaccine candidate.

Human Convalescent Sera

The neutralizing titer is the reciprocal of the highest sample dilutionthat protects at least 50% of cells from cytopathic effects. A panel of24 convalescent human serum samples were obtained from donors 18 to 70years of age (mean age, 45.8 years) who had recovered from SARS-CoV-2infection; samples were obtained at least 14 days after a polymerasechain reaction-confirmed diagnosis and after symptom resolution. Thedisease severities of these patients varied from non-symptomatic (n=3,13%), mild (n=8, 33%), moderate (n=10, 42%), or severe (n=3, 13%).

Neutralizing geometric mean titers (GMTs) in subgroups of the donorswere as follows: 40 for the 3 donors with non-symptomatic infections;91.9 for the 8 donors with mild infection; 160 for those with moderateinfection; and 226.3 in the 3 donors with severe infection. Each serumsample in the panel was from a different donor. Thus, most of the serumsamples were obtained from persons with moderate Covid-19. Theconvalescent serum samples were tested side by side as comparators withthe serum samples obtained from participants in this trial.

ELISA

We assessed binding antibody responses against the SARS-CoV-2 RBD and S1by using the enzyme-linked immunosorbent assay (ELISA).

Microneutralization Assay

We detected the SARS-CoV-2 specific neutralizing antibody in serum bymicroneutralization assay based on cytopathy observed in a biosafetylevel 3 laboratory (BSL-3) with SARS-CoV-2 virus strainBetaCoV/JS02/human/2020 (EPI_ISL_411952).

ELISpot

Specific T-cell responses against the peptides were assessed by using acommercial ex-vivo interferon-γ (INF-γ) enzyme-linked Immunospot(ELISpot) kit manufactured by Mabtech (Nacka Strand, Stockholm,Sweden)²². PBMCs were isolated from fresh blood samples, and stimulatedwith different overlapping peptide pools before the measurement. The S1peptide pool, which covers the N-terminal half of SARS-CoV-2 spike,including the RBD, and the S2 peptide pool, which covers the C-terminalof SARS-CoV-2 spike, which does not include the RBD were used in thisstudy²³. A peptide pool consisting of 32 MHC class I restricted viralpeptides from human Cytomegalovirus, Epstein-Barr virus and Influenzavirus (CEF peptide pool), was used to stimulate CD8+ T cells to assessgeneral T-cell reactivity (not specific to SARS-CoV-2²⁴)

Outcomes

The primary and secondary objectives of this trial were to evaluatesafety and immunogenicity of the candidate vaccine BNT162b1 in healthyChinese adults. The primary endpoints for safety evaluation were theincidence of solicited local reactions at the injection site or systemicadverse reactions within 14 days post vaccination, and adverse eventsfollowing the immunization until 28 days after receiving the boost dose.Any clinical laboratory abnormalities from baseline to 24 hours or 7days after vaccination, and any serious adverse event (SAE) thatoccurred were also recorded. The secondary endpoints for immunogenicitywere geometric mean titer (GMT), seroconversion rates, and fold increaseof virus-neutralizing antibody, and ELISA IgG antibodies binding to S1or RBD measured at days 8, 22 after each vaccination. Seroconversion isdefined as an increase by a factor of four or more in antibody titerover the baseline, or the lower limit value if the baseline titer isbelow the limit of detection. The serum dilution for ELISA started at1:100, while that for microneutralization assay started at 1:10.

Cellular immune responses in terms of the number of positive cells withinterferon gamma (IFN-γ) secretion among PBMCs at a concentration of1×10⁵/well at day 8 and 22 after the boost dose were explored as anexploratory endpoint.

Statistical Analysis

The total sample size in this study was 144 participants, 24participants of each age group was included in each treatment group.Based on an assumption of 8% of the adverse reaction occurrencepost-vaccination, the probability of observation of at least one eventin 24 participants in each dose group was 86.5%.

All randomized participants who received at least one dose of theinvestigational vaccine were included in the safety analysis. Safetyendpoints were described as frequencies (%) with 95% confidence interval(CI) of the adverse reactions or events during the observation period.We compared the proportions of the participants with adverse reactionsor events across the groups using Chi-square or Fisher exact. Allparticipants who received at least one vaccination and had results ofserologic measurements before or after vaccination were included in theimmunogenicity analysis. The immunological endpoints were descriptivelysummarized at the specified time points, and compared across the groups,using ANOVA for log-transformed antibody titres, or Wilcoxon rank-sumtest for non-normal data. The neutralising antibody responses of theparticipants in each dose group were compared with those of patients whohad PCR-confirmed SARS-CoV-2 infection. Any serologic values below thelower limit of detection were set to half of the value (1:50 for ELISAand 1:5 for microneutralization assay), while the values above thehighest dilution titer were assigned values of the highest dilution forcalculation. Pearson correlation analysis of the RBD or S1 specificELISA antibody and neutralising antibody was performed to assess therelationship between responses on different assays.

REFERENCES

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TABLE 24 Baseline characteristics of the participants, by age groups.Younger participants aged 18-55 years Older participants aged 65-85years Characteristic 10 μg 30 μg Placebo 10 μg 30 μg Placebo No. ofparticipants 24 24 24 24 24 24 Age, mean (SD), years 37.9 (9.6) 39.7(9.0) 42.0 (8.7) 70.5 (5.0) 68.5 (3.0) 70.7 (4.4) Sex (female) 12 (50%)12 (50%) 12 (50%) 12 (50%) 12 (50%) 12 (50%) Body-mass index, 24.7 (3.2)23.0 (2.7) 24.3 (3.4) 24.0 (3.0) 24.8 (2.9) 23.5 (2.5) kg/m² Medicalhistory or existing disorder Cardiac ischemia 2 (8%) 2 (8%) 2 (8%) 0 0 0Sinus bradycardia 0 2 (8%) 1 (4%) 0 0 0 Hyperuricemia 3 (13%) 1 (4%) 1(4%) 3 (13%) 2 (8%) 2 (8%) Nasopharyngitis 2 (8%) 0 0 0 0 0 Blood uricacid 2 (8%) 1 (4%) 1 (4%) 0 0 0 increased Hypertension 3 (13%) 0 1 (4%)12 (50%) 9 (38%) 7 (29%) Diabetes 0 0 0 1 (4%) 2 (8%) 1 (4%) Gastricinflammation 0 0 0 0 0 2 (8%) Others* 3 (13%) 5 (21%) 1 (4%) 3 (13%) 3(13%) 4 (17%) Data are mean (SD) or n (%). *“Others” includestonsillitis, helicobacter infection, human papilloma virus infection,periodontitis, electrocardiogram high voltage, lymphadenopathy, anemia,hepatic cyst, oropharyngeal discomfort, hyperthyroidism, noninfectivegingivitis, hyperlipaemia, benign prostatic hyperplasia, prostatitis,blindness unilateral, cerebral infarct, limb injury, deformity of spine,calculus urinary and lymphadenopathy.

TABLE 25A Solicited adverse reactions within 14 days post-vaccination,and unsolicited adverse reactions till day 43, by age groups. Youngerparticipants Older participants aged 18-55 years aged 65-85 yearsAdverse 10 μg 30 μg Placebo 10 μg 30 μg Placebo reactions (n = 24) (n =24) (n = 24) P value (n = 24) (n = 24) (n = 24) P value Solicitedadverse reactions within 14 days Any 21 (88%) 24 (100%)  4 (17%) <0.000121 (88%) 23 (96%) 2 (8%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2(8%) 0 0.3239 Injection-site adverse reactions Any 21 (88%) 24 (100%) 2(8%) <0.0001 18 (75%) 21 (88%) 0 <0.0001 Grade 3 0 0 0 — 0 0 0 — Pain 21(88%) 23 (96%) 2 (8%) <0.0001 16 (67%) 21 (88%) 0 <0.0001 Redness 6(25%) 8 (33%) 0 0.0059 3 (13%) 4 (17%) 0 0.1492 Swelling 5 (21%) 7 (29%)0 0.0137 0 5 (21%) 0 0.0091 Induration 0 3 (13%) 0 0.1018 0 1 (4%) 01.0000 Systemic adverse reactions Any 17 (71%) 22 (92%)  3 (13%) <0.00019 (38%) 19 (79%) 2 (8%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2(8%) 0 0.3239 Fever* 14 (58%) 21 (88%) 1 (4%) <0.0001 7 (29%) 19 (79%) 1(4%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2 (8%) 0 0.3239 Headache11 (46%) 19 (79%)  3 (13%) <0.0001 1 (4%) 2 (8%) 0 0.7682 Fatigue 12(50%) 16 (67%) 0 <0.0001 3 (13%) 8 (33%) 0 0.0045 Malaise 8 (33%) 9(38%) 0 0.0013 2 (8%) 4 (17%) 1 (4%) 0.4858 Joint pain 4 (17%) 10 (42%)1 (4%) 0.0067 0 1 (4%) 0 1.0000 Muscle pain 2 (8%) 10 (42%) 0 <0.0001 01 (4%) 0 1.0000 Chills 4 (17%) 7 (29%) 0 0.0118 1 (4%) 4 (17%) 0 0.1185Nausea 3 (13%) 3 (13%) 0 0.2330 0 0 0 — Anorexia 1 (4%) 4 (17%) 0 0.11850 3 (13%) 1 (4%) 0.3143 Diarrhea 2 (8%) 1 (4%) 1 (4%) 1.0000 0 0 01.0000 Vomiting 0 2 (8%) 0 0.3239 0 0 0 —

TABLE 25B (modified) Solicited adverse reactions within 14 dayspost-vaccination, and unsolicited adverse reactions until day 43, by agegroups including “placebo-corrected” AE rates. Younger participants aged18-55 years 10 μg 30 μg (minus (minus Adverse 10 μg placebo- 30 μgplacebo- Placebo reactions (n = 24) AEs) (n = 24) AEs) (n = 24) P valueSolicited adverse reactions within 14 days Any 21 (88%) 17 (71%) 24(100%) 20 (83%)  4 (17%) <0.0001 Grade 3 3 (13%) 3 (13%) 9 (38%) 9 (38%)0 0.0015 Injection site adverse reactions Any 21 (88%) 19 (79%) 24(100%) 22 (92%) 2 (8%) <0.0001 Grade 3 0 0 0 0 0 — Pain 21 (88%) 19(79%) 23 (96%) 21 (88%) 2 (8%) <0.0001 Redness 6 (25%) 6 (25%) 8 (33%) 8(33%) 0 0.0059 Swelling 5 (21%) 5 (21%) 7 (29%) 7 (29%) 0 0.0137Induration 0 0 3 (13%) 3 (13%) 0 0.1018 Systemic adverse reactions Any17 (71%) 14 (58%) 22 (92%) 19 (79%)  3 (13%) <0.0001 Grade 3 1 (4%) 1(4%) 4 (17%) 4 (17%) 0 0.0015 Fever* 14 (58%) 13 (54%) 21 (88%) 20 (83%)1 (4%) <0.0001 Grade 3 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.0015 Grade 3 by3 (13%) 3 (13%) 9 (38%) 9 (38%) 0 0.0015 NMPA criteria Headache 11 (46%)8 (33%) 19 (79%) 16 (67%)  3 (13%) <0.0001 Fatigue 12 (50%) 12 (50%) 16(67%) 16 (67%) 0 <0.0001 Malaise 8 (33%) 8 (33%) 9 (38%) 9 (38%) 00.0013 Joint pain 4 (17%) 3 (13%) 10 (42%) 9 (38%) 1 (4%) 0.0067 Musclepain 2 (8%) 2 (8%) 10 (42%) 10 (42%) 0 <0.0001 Chills 4 (17%) 4 (17%) 7(29%) 7 (29%) 0 0.0118 Nausea 3 (13%) 3 (13%) 3 (13%) 3 (13%) 0 0.2330Anorexia 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.1185 Diarrhea 2 (8%) 1 (4%) 1(4%) 0 1 (4%) 1.0000 Vomiting 0 0 2 (8%) 2 (8%) 0 0.3239 Unsolicitedadverse reactions within 28 days Any 9 (38%) 8 (33%) 10 (42%) 9 (38%) 1(4%) 0.0046 Fever^(†) 0 0 0 0 0 — Temperature 2 (8%) 2 (8%) 6 (25%) 6(25%) 0 0.0230 intolerance Injection site 3 (13%) 3 (13%) 4 (17%) 4(17%) 0 0.1492 discomfort Injection site 2 (8%) 2 (8%) 3 (13%) 3 (13%) 00.3580 pruritus Pain not at 1 (4%) 1 (4%) 1 (4%) 1 (4%) 0 1.0000injection site Dizziness 3 (13%) 3 (13%) 1 (4%) 1 (4%) 0 0.3142 Blooduric 1 (4%) 1 (4%) 1 (4%) 1 (4%) 0 1.0000 acid increased Olderparticipants aged 65-85 years 10 μg 30 μg (minus (minus Adverse 10 μgplacebo- 30 μg placebo- Placebo reactions (n = 24) AEs) (n = 24) AEs) (n= 24) P value Solicited adverse reactions within 14 days Any 21 (88%) 19(79%) 23 (96%) 21 (88%) 2 (8%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 00.3239 Injection site adverse reactions Any 18 (75%) 18 (75%) 21 (88%)21 (88%) 0 <0.0001 Grade 3 0 0 0 0 0 — Pain 16 (67%) 16 (67%) 21 (88%)21 (88%) 0 <0.0001 Redness 3 (13%) 3 (13%) 4 (17%) 4 (17%) 0 0.1492Swelling 0 0 5 (21%) 5 (21%) 0 0.0091 Induration 0 0 1 (4%) 1 (4%) 01.0000 Systemic adverse reactions Any 9 (38%) 7 (29%) 19 (79%) 17 (71%)2 (8%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 0 0.3239 Fever* 7 (29%) 6 (25%)19 (79%) 18 (75%) 1 (4%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 0 0.3239Grade 3 by 0 0 2 (8%) 2 (8%) 0 0.3239 NMPA criteria Headache 1 (4%) 1(4%) 2 (8%) 2 (8%) 0 0.7682 Fatigue 3 (13%) 3 (13%) 8 (33%) 8 (33%) 00.0045 Malaise 2 (8%) 1 (4%) 4 (17%) 3 (13%) 1 (4%) 0.4858 Joint pain 00 1 (4%) 1 (4%) 0 1.0000 Muscle pain 0 0 1 (4%) 1 (4%) 0 1.0000 Chills 1(4%) 1 (4%) 4 (17%) 4 (17%) 0 0.1185 Nausea 0 0 0 0 0 — Anorexia 0 −1  3(13%) 2 (8%) 1 (4%) 0.3143 Diarrhea 0 0 0 0 0 1.0000 Vomiting 0 0 0 0 0— Unsolicited adverse reactions within 28 days Any 4 (17%) 2 (8%) 9(38%) 7 (29%) 2 (8%) 0.0590 Fever^(†) 0 0 1 (4%) 1 (4%) 0 1.0000Temperature 0 0 4 (17%) 4 (17%) 0 — intolerance Injection site 2 (8%) 2(8%) 3 (13%) 3 (13%) 0 0.3580 discomfort Injection site 0 0 1 (4%) 1(4%) 0 1.0000 pruritus Pain not at 0 0 0 0 0 — injection site Dizziness0 0 3 (13%) 3 (13%) 0 0.1018 Blood uric 2 (8%) 0 1 (4%) −1  2 (8%)1.0000 acid increased Data are shown as number of participants withevent (%). Grade 3 was severe reaction (i.e., prevented activity). SAEs= Serious adverse events. A participant was only counted once in thespecific reaction category, also with more than one episode of theadverse reaction. Only unsolicited adverse reactions reported by two ormore participants were listed. *Those febrile participants were gradedaccording to the guidelines of Food and Drug Administration (FDA), theUnited States. Fever was also graded according to the grading guidelinesfor adverse events in vaccine clinical trials, issued by the NationalMedical Products Administration (NMPA), China, which defines grade 3fever as axillary temperature ≥38.5° C. ^(†)One participant experiencedgrade 3 fever accompanied with pain, itching and pruritus at theinjection site after the prime dose, and electively withdrew from theboost vaccination.

Example 33: Neutralization of SARS-CoV-2 Lineage B.1.1.7 Pseudovirus byBNT162b2 Vaccine-Elicited Sera

In September 2020, the SARS-CoV-2 variant B.1.1.7 was detected in theUnited Kingdom, and it subsequently increased in prevalence, showedenhanced transmissibility, and spread to other countries and continents.B1.1.7 has a series of mutations in its spike protein: AH69/V70, ΔY144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. One of these,N501Y, was of particular concern because it is located in the receptorbinding site; the spike with this mutation binds more tightly to itscellular receptor, ACE-2; and virus with this mutation has increasedhost range that includes mice. 19 pseudoviruses, each bearing aSARS-CoV-2 S with a different mutation found in circulating strains,were neutralized as efficiently as non-mutant pseudoviruses byBNT162b2-immune sera. The following study demonstrates that a virus withthe full set of mutations in the UK variant spike is also neutralizedefficiently by BNT162b2-immune sera.

We generated VSV-SARS-CoV-2-S pseudoviruses bearing the Wuhan referencestrain or lineage B.1.1.7 strain spike protein. Sera of 16 participantsin the previously reported trial (Sahin U. et al., medRxiv2020.12.09.20245175; doi: doi.org/10.1101/2020.12.09.20245175), drawnfrom eight younger (18-55 yrs) and eight older adults (56-85 yrs) 21days after the booster immunization with 30 μg BNT162b2, were tested forneutralization of SARS-CoV-2 Wuhan and lineage B.1.1.7 spike-pseudotypedVSV by a 50% pseudovirus neutralization assay (pVNT₅₀; FIG. 122 ). Theratio of the 50% neutralization GMT of the sera against the SARS-CoV-2lineage B.1.1.7 spike-pseudotyped VSV to that against the Wuhanreference spike-pseudotyped VSV was 0.79, indicating no biologicallysignificant difference in neutralization activity against the twopseudoviruses.

The preserved neutralization of pseudoviruses bearing the B.1.1.7 spikeby BNT162b2-immune sera suggests that the UK variant viruses will notescape BNT162b2-mediated protection. Furthermore, since there is goodconcordance between pseudotype neutralization and SARS-CoV-2neutralization assays, the use of a non-replicating pseudovirus systemis not expected to be a potential limitation of the work.

Materials and Methods VSV-SARS-CoV-2 S Variant Pseudovirus Generation

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-2spike (S) derived from either the Wuhan reference strain (NCBI Ref:43740568) or the variant of concern (VOC)-202012/01 (also known asSARS-CoV-2 lineage B.1.1.7) according to published pseudotypingprotocols (FIG. 123 ) (PMID: 21998709). In brief, HEK293T/17 monolayerstransfected to express SARS-CoV-2 S were inoculated with VSV-Gcomplemented VSVΔG vector. After incubation for 1 h at 37° C., theinoculum was removed. Cells were washed with PBS before mediumsupplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) wasadded to neutralize residual VSV-G complemented input virus.VSV-SARS-CoV-2-S pseudotype-containing medium was harvested 20 h afterinoculation, 0.2 m filtered and stored at −80° C. Prior to use in theneutralization test, the pseudovirus batches were titrated on Vero 76cells, and the percent infected cells determined by flow cytometry (FIG.124 ). Individual titers were calculated in transducing units (TU) permL. Production of the VSV-SARS-CoV-2-S pseudoviruses bearing the Wuhanreference strain or lineage B.1.1.7 strain spike protein yielded similartiters (Table 26).

TABLE 26 Titers of SARS-CoV-2 Wuhan reference strain and lineage B.1.1.7spike-pseudotyped VSV in transducing units (TU) per mL. VSV pseudovirusbearing Titer [TU/mL] Wuhan strain SARS-CoV-2 S 1.59 × 10⁵ LineageB.1.1.7 SARS-CoV-2 S 1.30 × 10⁵

Serum Specimens and Neutralization Assay

The immunization and serum collection regimen is illustratedschematically in FIG. 125 . For measuring neutralization titers, eachserum was 2-fold serially diluted in culture medium with the firstdilution of 1:20 (dilution range of 1:20 to 1:2560). VSV-SARS-CoV-2-Sparticles were diluted in culture medium to obtain 100 TU in the assay.Serum dilutions were mixed 1:1 with pseudovirus for 30 minutes at roomtemperature prior to addition to Vero 76 cell monolayers in 96-wellplates and incubation at 37° C. for 24 hours. Supernatants were removed,and the cells were lysed with luciferase reagent (Promega). Luminescencewas recorded, and neutralization titers were calculated in GraphPadPrism version 9 by generating a 4-parameter logistical (4PL) fit of thepercent neutralization at each serial serum dilution. The 50%pseudovirus neutralisation titre (pVNT₅₀) was reported as theinterpolated reciprocal of the dilution yielding a 50% reduction inluminescence. A table of the neutralization titers is provided (Table27). The ratio for each serum of the pVNT₅₀ against SARS-CoV-2 lineageB.1.1.7 and the Wuhan reference strain spike-pseudotyped VSV is plottedin FIG. 126 .

TABLE 27 pVNT₅₀ values of 16 BNT162b2 post-immunization sera againstSARS-CoV-2 Wuhan reference strain spike-pseudotype and lineage B.1.1.7spike-pseudotyped VSV. pVNT₅₀ pVNT₅₀ ratio Serum ID Wuhan ref. B.1.1.7(B.1.1.7/Wuhan ref.) 1 160 161.2 1.01 2 114.1 85.8 0.75 3 223.2 128.60.58 4 193 268.4 1.39 5 111.9 64.3 0.57 6 128 99.1 0.77 7 278.1 226.80.82 8 203.6 185 0.91 9 94.9 58.4 0.62 10 209.7 126.8 0.60 11 50.8 41.70.82 12 241.3 486.1 2.01 13 174 84.8 0.49 14 292.5 136.7 0.47 15 186.7121.6 0.65 16 86.3 116.2 1.35

Example 34: Exemplary Regimen for Administration of a SARS-CoV-2 RNAVaccine in Pregnant Women

The present Example describes an exemplary regimen for administration ofa SARS-CoV-2 RNA vaccine described herein (specifically, in thisExample, BNT162b2) in pregnant women (e.g., in healthy pregnant women 18years of age and older).

Pregnant women are at risk for acquiring SARS-CoV-2 infection andCOVID-19. Pregnancy may confer increased risk of severe COVID-19 becauseof physiological changes during pregnancy that can increasesusceptibility to respiratory infections and subsequent rapidprogression to respiratory failure. Additionally, pregnant women withCOVID-19 have been reported to have higher rates of preterm birth,cesarean delivery, fetal distress, and infants requiring neonatalintensive care.

The present Example describes certain protocols in accordance with whichBNT162b2 may be administered to pregnant women and/or to infants bornfrom such pregnant women, and also describes certain assessments thatmay be performed and/or results that may be achieved. For example, thisExample describes a study that will assess safety of BNT162b2 inpregnant women and their infants; it will also assess the immunogenicityof BNT162b2 in pregnant women, the transfer of antibody to theirinfants, and the kinetics of antibody transfer in the infant.

Among other things, the present Example describes a study that willassess the safety and tolerability of prophylactic BNT162b2 whenadministered to maternal participants 18 years of age or oldervaccinated at 24 to 34 weeks' gestation. Without wishing to be bound byany particular theory, the present Example proposes that vaccinationbeginning within this time period may provide particular advantages.Recognizing that proposals ranging from vaccination at any time duringpregnancy (see, for example, “Israel Recommends COVID Vaccination in AllStages of Pregnancy, Updating Guidelines” Haaretz Feb. 1, 2021) andothers have proposed refraining from vaccinating during pregnancy (see,for example, WHO Strategic Advisory Group recommendation), the presentExample describes a particular regimen in which pregnant mothers receivea first dose of vaccine between about 24 to about 34, or in someembodiments between about 27 to about 34 weeks of gestation, and asecond dose about 21 days later, ideally prior to delivery of the baby.

Without wishing to be bound by any particular theory, the presentExample proposes that vaccination according to this regimen may, forexample, reduce risk to the fetus as may result, for example, fromexposure to an immunized maternal immune response early in pregnancy.Furthermore, still without wishing to be bound by any particular theory,the present Example proposes that the provided vaccination schedule mayprovide particular benefits when at least two doses are administeredprior to delivery of the baby. Among other things, the present Exampleproposes that a provided regimen may provide a particularly beneficialrisk/benefit balance. Among other things, the present disclosure teachesthat benefits that may be provided by immunization of pregnant mothers,and particularly by such immunization in accordance with a regimendescribed in the present Example, may impart immunity to the baby that,in some embodiments, may carry past delivery, this reducing need forimmunization of the baby, at least for a period of days weeks, months,or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24or more months, or 1, 2, 3, 4, or 5 years) post-delivery. Thus, in someembodiments, as noted herein, infants born of mothers vaccinated duringpregnancy, e.g, according to a particular regimen as described herein,may not need further vaccination, or may need reduced vaccination (e.g.,lower doses and/or smaller numbers of administrations—e.g.,boosters—and/or lower overall exposure over a given period of time), fora period of time (e.g., as noted herein) after birth.

For example, in maternal participants receiving at least 1 dose of studyintervention from each vaccine group, the percentage of maternalparticipants reporting: (i) Local reactions for up to 7 days followingeach dose; (ii) Systemic events for up to 7 days following each dose(iii) AEs from Dose 1 through 1 month after Dose 2 (iv) SAEs from Dose 1through 1 month after delivery will be assessed. Alternatively oradditionally, in maternal participants complying with certain keyprotocol criteria (evaluable maternal participants) and no serologicalor virological evidence (up to 1 month after receipt of the second dose)of past SARS-CoV-2 infection: (v) GMR, estimated by the ratio of thegeometric mean of SARS-CoV-2 neutralizing titers in pregnant women tothose in nonpregnant women 1 month after Dose 2 may be assessed.

Still further alternatively or additionally, in maternal participantscomplying with the key protocol criteria (evaluable participants) and/orwith without or without (e.g., separately for those with and thosewithout, or independent of) serological or virological evidence (priorto 7 days after receipt of Dose 2) of past SARS-CoV-2 infection: (vi)100×(1−IRR) [ratio of active vaccine to placebo] may be assessed.

Yet further alternatively or additionally, one or more of the followingmay be assessed:

-   -   In maternal participants complying with the key protocol        criteria (evaluable maternal participants) from each vaccine        group: (a) GMCs/GMTs, at baseline (before Dose 1), 2 weeks after        Dose 2, 1 month after Dose 2, and 6 months after delivery (b)        GMFRs from baseline through 2 weeks after Dose 2, 1 month after        Dose 2, and 6 months after delivery;    -   In infants born to maternal participants receiving at least 1        dose of study intervention from each vaccine group, the        percentage of infants with: (a) Specific birth outcomes (b) AEs        from birth through 1 month of age (c) SAEs and AESIs (major        congenital anomalies, developmental delay) through 6 months of        age;    -   In infants born to evaluable maternal participants from each        vaccine group: (a) GMCs and GMFRs, at birth and 6 months after        delivery;    -   In maternal participants who received BNT162b2 (at initial        randomization and at 1 month after delivery): (a) Incidence per        1000 person-years of follow-up;    -   In maternal participants who received BNT162b2 at initial        randomization and without evidence of prior SARS-CoV-2        infection: (a) Incidence per 1000 person-years of follow-up    -   In each subset of evaluable maternal participants from each        vaccine group with: (a) Confirmed COVID-19 (b) Confirmed severe        COVID-19 (c) SARS-CoV-2 infection but no confirmed COVID-19 (d)        GMCs/GMTs and GMFRs at baseline, 1 month after Dose 2, and 6        months after delivery;    -   In evaluable maternal participants: (a) GMCs/GMTs at baseline        and before Dose 2 (b) GMFRs from baseline to before Dose 2;    -   In infants born to maternal participants from each vaccine        group, based on the breastfeeding status: (a) GMCs and GMFRs, at        birth and 6 months after delivery;    -   In infants born to maternal participants receiving at least 1        dose of study intervention from each vaccine group, based on the        breastfeeding status, the percentage of infants with: (a) AEs        from birth through 1 month of age (b) SAEs and AESIs (major        congenital anomalies, developmental delay) through 6 months of        age;    -   In infants born to maternal participants from each vaccine        group: (a) Incidence rate of infant participants with confirmed        COVID-19;    -   In infants born to maternal participants from each vaccine        group: (a) Incidence rate of MIS-C.

In some embodiments, a first dose will be administered to pregnant womenduring their 27 to 34 weeks of gestation, followed by a second doseapproximately 21 days later. In some embodiments, a first dose will beadministered to pregnant women during their 24 to 34 weeks of gestation,followed by a second dose approximately 21 days later. In someembodiments, participant mothers are assessed for a period of time up toabout 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18 months or more (e.g.,after initiation of the study, after administration of the first dose,after administration of the second dose, and/or after birth of theinfant).

Is some embodiments, an infant born to a mother to whom one or more(e.g., two) vaccine doses have been administered (e.g., to whom twodoses were administered during gestation) is assessed for a period oftime up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 months or more (e.g., after initiation of the study, afteradministration of the first dose, after administration of the seconddose, and/or after birth of the infant).

In some embodiments, a dose will be 30 ug of BNT162b2 as describedherein.

In some embodiments, assessment of vaccine performance are made inpopulations of pregnant women of any age, or within a particular agerange (e.g., equal to or above 18 years of age). In some embodiments,assessment of vaccine performance are made in populations of womencarrying singleton pregnancies.

In some embodiments, gestational age is assessed by one or more of lastmenstrual period, ultrasound examination, physical examination, and/orcombinations thereof. In some embodiments, gestational age is determinedby ultrasound. In some embodiments, gestational age is determined byconsideration of two or more assessments (e.g., two or more ultrasoundsperformed at different times, such as in different trimesters, of thepregnancy). In some embodiments, assessment of vaccine performance ismade in populations characterized by one or more of: ultrasoundexamination performed at at least 18 weeks of pregnancy with nosignificant fetal abnormalities observed (e.g., upon assessment by alicensed investigator); documented negative HIV, syphilis, and/or HBVtests or combinations thereof; prepregnancy BMI of ≤40 kg/m².

In some embodiments, assessment of vaccine performance is made inpopulations that do not include subjects characterized by one or moreof: suffering from a medical or psychiatric condition that may increasethe risk of vaccination or otherwise, in the reasonable judgement of alicensed investigator make the subject inappropriate for receipt of thevaccine; prevopis clinical or micobiological diagnosis of COVID-19;history of severe adverse reaction associated with a vaccine and/orsever allergic reaction (e.g., anaphylaxis) to any component of thevaccine; known or suspected immunodeficiency; bleeding diathesis orcondition associated with prolonged bleeding, gestational hypertensionor preeclampsia-eclampsia, placental abnormality, polyhydramnios oroligohydramnios, significant bleeding or blood clotting disorder,gestational diabetes, one or more signs of premature labor with thecurrent pregnancy or having ongoing intervention (medical/surgical) inthe current pregnancy to prevent preterm birth, prior stillbirth orneonatal death, prior low birth weight or preterm delivery, priorhistory of at least 3 miscarriages, prior pregnancies numbering greaterthan 5, or previous infant with a known genetic disorder or majorcongenital anomaly, previous vaccination with any coronavirus vaccine,receipt of medications intended to prevent COVID-19, receipt ofblood/plasma products or immunoglobulin from 60 days beforeadministration of study intervention or planned receipt through delivery(with 1 exception, anti-D immunoglobulin (eg, RhoGAM), which can begiven at any time), current alcohol abuse or illicit drug use,participants who receive treatment with immunosuppressive therapy(including cytotoxic agents or systemic corticosteroids, eg, for canceror an autoimmune disease, or planned receipt through the postvaccinationblood draw), participation in other studies involving study interventionwithin 28 days prior to study entry and/or during study participation,previous participation in other studies involving study interventioncontaining LNPs, current febrile illness, current symptoms of COVID-19infection, Receipt of any seasonal or pandemic influenza vaccine in theprevious 14 days, anticipated receipt of any seasonal or pandemicinfluenza vaccine in the 7 days after study intervention administration,receipt of a tetanus-, diphtheria-, and/or pertussis-containing vaccinein the previous 14 days, anticipated receipt of a tetanus-, diphtheria-,and/or pertussis-containing vaccine in the 7 days after studyintervention administration, receipt of short-term (<14 days) systemiccorticosteroids less than 28 days prior to dosing (inhaled/nebulized,intra-articular, intrabursal, or topical (skin or eyes) corticosteroidsare permitted).

In some embodiments, a mother vaccinated as described herein may betaking or may begin taking, for example, an antipyretic or other painmedication to treat symptoms associated with the vaccination.Alternatively or additionally, in some embodiments, a mother may betaking or may begin taking a medication required for treatment of apreexisting stable condition and/or an inhaled, topical or localizedinjection of corticosteroids.

In some embodiments, a mother vaccinated as described herein may begiven one or more antenatal corticosteroids, in particular if thepregnancy is at risk for preterm delivery. In some embodiments, thecorticosteroid is a glucocorticoid. In some embodiments, thecorticosteroid is betamethasone or progesterone, or a mixture thereof.

In some embodiments, vaccination as described in this Example reducesincidence of COVID-19 disease (and/or documented SARS-CoV-2 infection),or of severe COVID-19 disease, in mothers and/or infants born to them,for example relative to that observed in a comparable unvaccinated(e.g., having received placebo) population. In some embodiments, amother is considered to have COVID-19 disease if least 1 of symptom ofCOVID-19 disease (fever; new or increased cough; new or increasedshortness of breath; new or increased muscle pain; new loss of taste orsmell; sore throat; diarrhea; vomiting; and/or in some embodimentsfatigue, headache, nasal congestion or runny nose, nausea) is presentand a SARS-CoV-2 NAAT-positive test is obtained during, or within 4 daysbefore or after, the symptomatic period, either at the centrallaboratory or at a local testing facility (using an acceptable test). Insome embodiments, a mother is considered to have severe COVID-19 diseaseif she has confirmed COVID-19 and one or more of the following ispresent:

-   -   Clinical signs at rest indicative of severe systemic illness        (RR≥30 breaths/min, HR≥125 beats/min, SpO≤593% on room air at        sea level, or PaO2/FiO2<300 mm Hg);    -   Respiratory failure (defined as needing high-flow oxygen,        noninvasive ventilation, mechanical ventilation, or ECMO);    -   Evidence of shock (SBP<90 mm Hg, DBP<60 mm Hg, or requiring        vasopressors);    -   Significant acute renal, hepatic, or neurologic dysfunction*;    -   Admission to an ICU;    -   Death.

In some embodiments, an infant is considered to have COVID-19 disease ifat least one symptom (fever, new or increased cough, new or increasedshortness of breath, diarrhea, vomiting; and/or in some embodiments oneor more of nasal congestion or runny nose, poor appetite or poorfeeding, abdominal pain/colic) is present and a SARS-COV-2 NAAT-positivetest result is obtained during, or within 4 days before or after, thesymptomatic period, either at the central laboratory or at a localtesting facility (using an acceptable test). In some embodiments, aninfant is considered to have severe COVID-19 disease if she hasconfirmed COVID-19 and one or more of the following is present:

(i) Clinical signs at rest indicative of severe systemic illness:

-   -   RR (breaths/min): >50 from birth to 1 week of age, ≥40 from 1        week to 1 month of age, ≥34 from 1 month to 6 months of age;    -   HR (beats/min): >180;    -   SpO2≤92% on room air or >50% FiO2 to maintain ≥92%, or        PaO2/FiO2<300 mm Hg24;        (ii) Respiratory failure (defined as needing high-flow oxygen        including nasal CPaP/BiPaP, noninvasive ventilation, mechanical        ventilation, or ECMO);        (iii) Evidence of shock or cardiac failure:    -   SBP (mm Hg) (<5th percentile for age):        -   +<65 from birth to 1 week of age, <75 from 1 week to 1 month            of age, <100 from 1 month to 6 months of age;

OR

-   -   Requiring vasoactive drugs to maintain BP in the normal range;        (iv) Significant acute renal failure: serum creatinine>2 times        ULN for age or 2-fold increase in baseline creatinine;        (v) Significant GI/hepatic failure: total bilirubin>4 mg/dL or        ALT 2 times ULN for age;        (vi) Significant neurologic dysfunction: Glasgow Coma Scale        score<11 or acute change in mental status with a decrease in        Glasgow Coma Scale score≥3 points from abnormal baseline;        (vii) Admission to an ICU;        (viii) Death.

In some embodiments, incidence of multisystem inflammatory syndrome isnot significantly increased (e.g., relative to a comparable populationwhose mothers were not vaccinated, and/or who were not vaccinatedaccording to a protocol as described herein) in infants whose motherswere vaccinated as described herein. In some embodiments, an infant isconsidered to have multisystem inflammatory syndrome if:

-   -   the infant presents with fever (≥38.0° C. for ≥24 hours or        report of subjective fever lasting ≥24 hours); AND    -   there is laboratory evidence of inflammation (based on local        laboratory ranges) including, but not limited to, 1 or more of        the following: an elevated CRP, ESR, fibrinogen, procalcitonin,        D-dimer, ferritin, LDH, or IL-6, elevated neutrophils, reduced        lymphocytes, and low albumin; AND    -   there is evidence of clinically severe illness requiring        hospitalization (definition as noted above for severe disease),        with multisystem (2) organ involvement:        -   Cardiac (eg, shock, elevated troponin, elevated BNP,            abnormal echocardiogram, arrhythmia);        -   Renal (eg, acute kidney injury or renal failure);        -   Respiratory (eg, pneumonia, ARDS, pulmonary embolism);        -   Hematologic (eg, elevated D-dimers, thrombophilia, or            thrombocytopenia);        -   GI/hepatic (eg, elevated bilirubin, elevated liver enzymes,            or diarrhea);        -   Dermatologic (eg, rash, mucocutaneous lesions);        -   Neurological (eg, CVA, aseptic meningitis, encephalopathy);            AND    -   there is no alternative plausible diagnoses; AND    -   the infant is determined to be positive for current or recent        SARS-CoV-2 infection by RT-PCR, serology, or antigen test; OR    -   the infant has had COVID-19 exposure within the 4 weeks prior to        the onset of symptoms.

In some embodiments, vaccination of mothers as described herein does notmaterially increase incidence of preterm delivery of infant morbidity.

In some embodiments, incidence of COVID-19 disease (and/or documentedSARS-CoV-2 infection) in infants whose mothers were vaccinated asdescribed herein is reduced relative to that of infants whose motherswere not so vaccinated. In some embodiments, incidence of COVID-19disease (and/or documented SARS-CoV-2 infection) in infants whosemothers were vaccinated as described herein is comparable to that ofinfants who were directly vaccinated after their delivery.

In some embodiments, vaccination as described herein achieves one ormore of the following Primary or Secondary Outcome measures:

Primary Outcome Measures:

1. Percentage of maternal participants reporting: Local reactionsPain at the injection site, redness, and swelling as self-reported onelectronic diaries[Time Frame: For 7 Days after Dose 1 and Dose 2]2. Percentage of maternal participants reporting systemic eventsFever, fatigue, headache, chills, vomiting, diarrhea, new or worsenedmuscle pain, and new or worsened joint pain asself-reported on electronic diaries.[Time Frame: For 7 days after Dose 1 and Dose 2]3. Percentage of maternal participants reporting adverse eventsAs elicited by investigational site staff[Time Frame: From Dose 1 through 1 month after Dose 2]4. Percentage of maternal participants reporting serious adverse eventsAs elicited by investigational site staff[Time Frame: From Dose 1 through 6 months after delivery]5. Demonstrate non inferiority of immune response in pregnant womencompared to nonpregnant female participantsfrom the C4591001 study without evidence of past SARS-CoV-2 infection.GMR, estimated by the ratio of the geometric mean of SARS CoV 2neutralizing titers in pregnant women to those innonpregnant female participants[Time Frame: 1 month after Dose 2]6. Demonstrate non inferiority of immune response in pregnant womencompared to nonpregnant female participantsfrom the C4591001 study with and without evidence of prior SARS-CoV-2infectionGMR, estimated by the ratio of the geometric mean of SARS CoV 2neutralizing titers in pregnant women to those innonpregnant female participants[Time Frame: 1 month after Dose 2]

Secondary Outcome Measures:

7. Evaluate efficacy against confirmed COVID 19 in participants withoutevidence of infection prior to vaccination1000 person years of follow-up[Time Frame: 7 days after Dose 2]8. Evaluate efficacy against confirmed COVID 19 in participants withoutevidence of prior infection.1000 person years of follow-up[Time Frame: 7 days after Dose 2]

Example 35: Neutralization of SARS-CoV-2 Lineage B.1.1.298 (DanishStrain; a.k.a., SARS-CoV-2/Hu/DK/CL-5/1 (Cluster 5)) and B.1.351 (SouthAfrican Strain; a.k.a., 20H/501Y.V2 (501.V2)) Pseudovirus by BNT162b2Vaccine-Elicited Human Sera

Sera of 12 younger adult participants in the previously reported Germanphase 1/2 trial drawn at 7 or 21 days after the booster immunizationwith 30 μg BNT162b2, were tested for neutralization of SARS-CoV-2 WuhanHu-1 (reference), South African lineage B.1.351 (SA-strain), and Danishmink-related lineage B.1.1.298 (DNK-strain) spike protein pseudotypedVSV by a 50% neutralization assay (pVNT50). The SA-strain spike proteincarries the following amino acid changes compared to the Wuhanreference: L18F, D80A, D215G, ΔL242-244, R246I, K417N, E484K, N501Y,D614G, A701V. The DNK-strain spike protein carries the following aminoacid changes compared to the Wuhan reference: Y453F, D614G, I692V,M1229I.

BNT162b2-immune sera neutralized the DNK-strain pseudovirus almost asefficiently as the SARS-CoV-2 Wuhan Hu-1 pseudotyped reference. Adecrease (5-fold) in neutralizing titers was measured against theSARS-CoV-2 lineage B.1.351 pseudovirus when comparing the titers to theWuhan Hu-1 pseudotyped reference. Importantly, all testedBNT162b2-immune sera were still able to neutralize with no completeescape being noted (FIG. 127 ).

Materials and Methods:

A recombinant replication-deficient VSV vector that encodes greenfluorescent protein (GFP) and luciferase (Luc) instead of theVSV-glycoprotein (VSV-G) was pseudotyped with Wuhan-Hu-1 isolateSARS-CoV-2 spike (S) (GenBank: QHD43416.1), a variant harbouring fourmutations found in the S protein of the Danish mink-related lineageB.1.1.298 (Y453F, D614G, I692V, M1229I), or variants harbouring tenmutations (L18F, D80A, D215G, R246I, Δ242/243/244, K417N, E484K, N501Y,D614G, A701V) found in the South African lineage B.1.351 S proteinaccording to published pseudotyping protocols. In brief, HEK293T/17monolayers transfected to express the respective SARS-CoV-2 S truncatedof the C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S(CΔ19)) wereinoculated with VSVΔG-GFP/Luc vector. After incubation for 1 h at 37°C., the inoculum was removed, and cells were washed with PBS beforemedium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast)was added to neutralise residual input virus. VSV-SARS-CoV-2pseudovirus-containing medium was collected 20 h after inoculation,0.2-m-filtered and stored at −80° C.

For pseudovirus neutralisation assays, 40,000 Vero 76 cells were seededper 96-well. Sera were serially diluted 1:2 in culture medium startingwith a 1:10 dilution (dilution range of 1:10 to 1:2,560).VSV-SARS-CoV-2-S pseudoparticles were diluted in culture medium for afluorescent focus unit (ffu) count in the assay of ^(˜)1,000 TU in theassay. Serum dilutions were mixed 1:1 with pseudovirus for 30 minutes atroom temperature prior to addition to Vero 76 cell monolayers in 96-wellplates and incubation at 37° C. for 24 hours. Supernatants were removed,and the cells were lysed with luciferase reagent (Promega). Luminescencewas recorded, and neutralisation titers were calculated in GraphPadPrism version 9 by generating a 4-parameter logistical (4PL) fit of thepercent neutralisation at each serial serum dilution. The 50%pseudovirus neutralisation titre (pVNT50) was reported as theinterpolated reciprocal of the dilution yielding a 50% reduction inluminescence.

Example 36: Neutralization of N501Y Mutant SARS-CoV-2 by BNT162b2Vaccine-Elicited Sera

Rapidly spreading variants of SARS-CoV-2 have arisen in the UnitedKingdom and South Africa (Volz E. et al. Report 42—Transmission ofSARS-CoV-2 Lineage B.1.1.7 in England: Insights from linkingepidemiological and genetic data.www.imperial.ac.uk/mrc-global-infectious-disease-analysis/covid-19/report-42-sars-cov-2-variant/;Tegally H. et al. Emergence and rapid spread of a new severe acuterespiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage withmultiple spike mutations in South Afric. medRxiv 2020.doi.org/10.1101/2020.12.21.20248640). These variants have multiplemutations in their S glycoproteins, which are key targets of virusneutralizing antibodies. These rapidly spreading variants share thespike N501Y substitution. This mutation is of particular concern becauseit is located in the viral receptor binding site for cell entry,increases binding to the receptor (angiotensin converting enzyme 2), andenables the virus to expand its host range to infect mice (Gu H. et al.Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.Science 2020; 369:1603-7; Chan K. K. et al. An engineered decoy receptorfor SARS-CoV-2 broadly binds protein S sequence variants. Cold SpringHarbor Laboratory 2020.doi: 10.1101/2020.10.18.344622).

We generated an isogenic Y501 SARS-CoV-2 on the genetic background ofthe N501 clinical strain USA-WA1/2020, which also provided the geneticbackground of the BNT162b2-encoded spike antigen. Sera of 20participants in the previously reported trial (Walsh E. E. et al. Safetyand Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N EnglJ Med 2020; Polack F. P. et al. Safety and efficacy of the BNT162b2 mRNACovid-19 vaccine. N Eng. J Med 2020. DOI: 10.1056/NEJMoa2034577), drawn2 or 4 weeks after immunization with two 30-μg doses of BNT162b2 spacedthree weeks apart, were tested for neutralization of N501 and Y501viruses by a 50% plaque reduction neutralization assay (PRNT₅₀; FIG. 128). The ratio of the 50% neutralization GMT of the sera against the Y501virus to that against the N501 virus was 1.46, indicating no reductionin neutralization activity against the virus bearing the Y501 spike.

Materials and Methods Construction of Isogenic Viruses

We prepared an isogenic pair of SARS-CoV-2 containing the N501 or Y501spike protein (FIG. 129 ). The N501Y mutation was generated by an A-to-Tsubstitution at nucleotide 23,063 of the viral genome using aninfectious cDNA clone of clinical strain WA1 (2019-nCoV/USA_WA1/2020)(Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe2020; 27:841-8 e3). Following a previously reported mutagenesis protocol(Plante J. A. et al. Spike mutation D614G alters SARS-CoV-2 fitness.Nature 2020), we recovered N501 and Y501 viruses with titers of >10⁷plaque-forming units (PFU) per ml. The two viruses developed similarplaque morphologies on Vero E6 cells (FIG. 130 ).

Serum Specimens and Neutralization Assay

The immunization and serum collection regimen is illustratedschematically in FIG. 131 . For measuring neutralization titers, eachserum was 2-fold serially diluted in culture medium with the firstdilution of 1:40 (dilution range of 1:40 to 1:1280). The diluted serumwas incubated with 100 PFU of N501 or Y501 virus at 37° C. for 1 h,after which the serum-virus mixtures were inoculated onto Vero E6 cellmonolayer in 6-well plates. A conventional (non-fluorescent) plaquereduction neutralization assay was performed to quantify theserum-mediated virus suppression as previously reported (Muruato A. E.et al. A high-throughput neutralizing antibody assay for COVID-19diagnosis and vaccine evaluation. Nat Commun 2020; 11:4059). A minimalserum dilution that suppressed >50% of viral plaques is defined asPRNT₅₀. A table of the neutralization titers is provided (Table 28). Theratio for each serum of the PRNT₅₀ against N501 and Y501 virus isplotted in FIG. 132 .

TABLE 28 PRNT₅₀ values of 20 BNT162b2 post-immunization sera againstN501 and Y501 SARS-CoV-2. PRNT₅₀ PRNT₅₀ ratio Serum ID N501 Y501(Y501/N501) 1 160 640 4 2 160 320 2 3 320 640 2 4 80 160 2 5 160 160 1 6320 320 1 7 640 640 1 8 160 160 1 9 640 640 1 10 640 1280 2 11 160 640 412 320 320 1 13 640 1280 2 14 640 320 0.5 15 320 640 2 16 320 640 2 17640 640 1 18 640 1280 2 19 640 640 1 20 640 640 1

Example 37: Neutralization of Spike 69/70 Deletion, E484K, and N501YSARS-CoV-2 by BNT162b2 Vaccine-Elicited Sera

Rapidly spreading variants of SARS-CoV-2 have arisen in the UnitedKingdom (UK), South Africa (SA), and other regions (Volz E. et al. CMe.Report 42—Transmission of SARS-CoV-2 Lineage B.1.1.7 in England:Insights from linking epidemiological and genetic data.wwwimperialacuk/mrc-global-infectious-disease-analysis/covid-19/report-42-sars-cov-2-variant/2021;Tegally H. et al. e. Emergence and rapid spread of a new severe acuterespiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage withmultiple spike mutations in South Africa medRxiv 2020.doi.org/10.1101/2020.12.21.20248640). These variants have multiplemutations in their spike glycoproteins, which are key targets of virusneutralizing antibodies. The emerged spike mutations have raisedconcerns of vaccine efficacy against these new strains. The goal of thisstudy is to examine the effect of several key spike mutations from theUK and SA strains on BNT162b2 vaccine-elicited neutralization.

We engineered three SARS-CoV-2s containing key spike mutations from thenewly emerged United Kingdom (UK) and South African (SA) variants: N501Yfrom UK and SA; 69/70−deletion+N501Y+D614G from UK; andE484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs)of twenty BTN162b2-vaccinated human sera against the three mutantviruses were 0.81- to 1.46-fold of the GMTs against parental virus,indicating small mutational effects on neutralization by sera elicitedby two BNT162b2 doses.

Using an infectious cDNA clone of SARS-CoV-2 (Xie X. et al. AnInfectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8e3), we engineered three spike mutant viruses on the genetic backgroundof clinical strain USA-WA1/2020 (FIG. 133 ). (i) Mutant N501Y viruscontains the N501Y mutation that is shared by both the UK and SAvariants. This mutation is located in the viral receptor binding domain(RBD) for cell entry, increases binding to the receptor (angiotensinconverting enzyme 2), and enables the virus to expand its host range toinfect mice (Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. CellHost Microbe 2020; 27:841-8 e3; Wrapp D. et al. Cryo-EM structure of the2019-nCoV spike in the prefusion conformation. Science 2020;367:1260-3). (ii) Mutant Δ69/70+N501Y+D614G virus contains twoadditional changes from the UK variants: amino acid 69 and 70 deletion(A69/70) and D614G substitution. Amino acids 69 and 70 are located inthe N-terminal domain of the spike S1 fragment; deletion of theseresidues may allosterically change the conformation of spike (Wrapp D.et al. Cryo-EM structure of the 2019-nCoV spike in the prefusionconformation. Science 2020; 367:1260-3). The D614G mutation is dominantin circulating strains around the world (Plante J A et al. Spikemutation D614G alters SARS-CoV-2 fitness. Nature 2020; Korber B. et al.Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G IncreasesInfectivity of the COVID-19 Virus. Cell 2020). (iii) MutantE484K+N501Y+D614G virus contains the E484K substitution, which is alsolocated in the viral RBD. The E484K substitution alone confersresistance to several monoclonal antibodies (Ku Z. et al. Moleculardeterminants and mechanism for antibody cocktail preventing SARS-CoV-2escape. Nat Commun 2021; 12:469; Baum A. et al. Antibody cocktail toSARS-CoV-2 spike protein prevents rapid mutational escape seen withindividual antibodies. Science 2020; 369:1014-8). Compared with thewild-type USA-WA1/2020 strain, the three mutant viruses showed similarplaque morphologies on Vero E6 cells (FIG. 134 ).

We tested a panel of human sera from twenty participants in thepreviously reported clinical trial (Walsh E E et al. Safety andImmunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N Engl JMed 2020; Polack F P et al. Safety and Efficacy of the BNT162b2 mRNACovid-19 Vaccine. N Engl J Med 2020), drawn 2 or 4 weeks afterimmunization with two 30-μg doses of BNT162b2 spaced three weeks apart(FIG. 135 ). Each serum was tested for neutralization of wild-typeUSA-WA1/2020 strain and the three mutant viruses by a 50% plaquereduction neutralization assay (PRNT₅₀; Tables 29 and 30).

TABLE 29 PRNT₅₀s of twenty BNT162b2 post-immunization sera againstwild-type (USA-WA1/2020) and mutant N501Y SARS-CoV-2s PRNT₅₀ PRNT₅₀ratio Serum ID WT N501Y (N501Y/WT) 1 160 640 4 2 160 320 2 3 320 640 2 480 160 2 5 160 160 1 6 320 320 1 7 640 640 1 8 160 160 1 9 640 640 1 10640 1280 2 11 160 640 4 12 320 320 1 13 640 1280 2 14 640 320 0.5 15 320640 2 16 320 640 2 17 640 640 1 18 640 1280 2 19 640 640 1 20 640 640 1

TABLE 30 PRNT₅₀s of twenty BNT162b2 post-immunization sera againstwild-type (USA-WA1/2020), Δ69/70 + N501Y + D614G, and E484K + N501Y +D614G SARS-CoV-2s PRNT₅₀ PRNT₅₀ ratio Δ69/70 + E484K + Δ69/70 + E484K +Serum N501Y + N501Y + N501Y + N501Y + ID WT D614G D614G D614G/WTD614G/WT 1 320 640 320 2 1 2 160 160 80 1 0.5 3 640 1280 640 2 1 4 160160 80 1 0.5 5 320 320 320 1 1 6 640 640 640 1 1 7 640 1280 320 2 0.5 8320 320 160 1 0.5 9 1280 1280 1280 1 1 10 640 1280 640 2 1 11 320 320320 1 1 12 640 1280 320 2 0.5 13 1280 2560 1280 2 1 14 320 320 320 1 115 320 640 320 2 1 16 640 640 640 1 1 17 640 1280 640 2 1 18 320 640 3202 1 19 640 640 320 1 0.5 20 640 1280 640 2 1

All sera showed equivalent neutralization titers between the wild-typeand mutant viruses, with differences of ≤4 fold (FIG. 136 ). Notably,ten out of the twenty sera had neutralization titers against mutantΔ69/70+N501Y+D614G virus that were twice their titers against thewild-type virus (FIG. 136 b ), whereas six out of the twenty sera hadneutralization titers against mutant E484K+N501Y+D614G virus that werehalf their titers against the wild-type virus (FIG. 136 c ). The ratiosof the neutralization GMTs of the sera against the N501Y,Δ69/70+N501Y+D614G, and E484K+N501Y+D614G viruses to their GMTs againstthe USA-WA1/2020 virus were 1.46, 1.41, and 0.81, respectively (FIG. 137).

Consistent with other recent reports of the neutralization of variantSARS-CoV-2 or corresponding pseudoviruses by convalescent orpost-immunization sera (Wibmer C K et al. SARS-CoV-2 501Y.V2 escapesneutralization by South African COVID-19 donor plasma. bioRxiv 2021:doi:doi.org/10.1101/2021.01.18.427166; Wang Z. et al. mRNA vaccine-elicitedantibodies to SARS-CoV-2 and circulating variants. bioRxiv 2021:doi:doi.org/10.1101/2021.01.15.426911), the neutralization GMT of the serumpanel against the virus with three mutations from the SA variant(E484K+N501Y+D614G) was slightly lower than the neutralization GMTsagainst the N501Y virus or the virus with three mutations from the UKvariant (Δ69/70+N501Y+D614G). However, the magnitude of the differencesin neutralization GMTs against any of the viruses in this study wassmall, as compared to the 4-fold differences inhemagglutination-inhibition titers that have been used to signalpotential need for a strain change in influenza vaccines (Smith D J etal. Mapping the antigenic and genetic evolution of influenza virus.Science 2004; 305:371-6).

Methods Construction of Isogenic Viruses.

Three recombinant SARS-CoV-2 mutants (N501Y, Δ69/70−N501Y+D614G,E484K+N501Y+D614G in spike protein) were prepared on the geneticbackground of an infectious cDNA clone derived from clinical strain WA1(2019-nCoV/USA_WA1/2020) (Xie X. et al. An Infectious cDNA Clone ofSARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3) by following thePCR-based mutagenesis protocol as reported previously (Plante J A et al.Spike mutation D614G alters SARS-CoV-2 fitness. Nature 2020). Thefull-length infectious cDNAs were in vitro ligated and used as templatesto transcribe full-length viral RNA. Mutant viruses (P0) were recoveredon day 2 from Vero E6 cells after electroporation of the in vitro RNAtranscripts. P1 viruses were harvested as stocks by passaging the P0virus once on Vero E6 cells. The titers of P1 viruses were determined byplaque assay on Vero E6 cells. The genome sequences of the P1 viruseswere validated by Sanger sequencing. The detailed protocol was recentlyreported (Xie X. et al. Engineering SARS-CoV-2 using a reverse geneticsystem. Nature Protocols 2021:doi.org/10.1038/s41596-021-00491-8).

Serum Specimens and Neutralization Assay.

The immunization and serum collection regimen are illustratedschematically in FIG. 135 . A conventional (non-fluorescent) plaquereduction neutralization assay was performed to quantify theserum-mediated virus suppression as previously reported (Muruato A E etal. A high-throughput neutralizing antibody assay for COVID-19 diagnosisand vaccine evaluation. Nat Commun 2020; 11:4059). Briefly, each serumwas 2-fold serially diluted in culture medium with the first dilution of1:40 (dilution range of 1:40 to 1:1280). The diluted sera were incubatedwith 100 plaque-forming units of wild-type or mutant viruses at 37° C.for 1 h, after which the serum-virus mixtures were inoculated onto VeroE6 cell monolayer in 6-well plates. After 1 h of infection at 37° C., 2ml of 2% Seaplaque agar (Lonza) in Dulbecco's modified Eagle medium(DMEM) containing 2% fetal bovine serum (FBS) and 1%penicillin/streptomycin (P/S) was added to the cells. After 2 days ofincubation, 2 ml of 2% Seaplaque agar (Lonza) in DMEM containing 2% FBS,1% P/S and 0.01% neutral red (Sigma) were added on top of the firstlayer. After another 16 h of incubation at 37° C., plaque numbers werecounted. The minimal serum dilution that inhibits 50% of plaque countsis defined as the 50% plaque reduction neutralization titer (PRNT₅₀).Each serum was tested in duplicates. The PRNT₅₀ assay was performed atthe biosafety level-3 facility at the University of Texas MedicalBranch.

Example 38: Neutralizing Activity of BNT162b2-Elicited Serum

New, highly transmissible SARS-CoV-2 variants that were first detectedin the United Kingdom (B.1.1.7 lineage), South Africa (B.1.351 lineage),and Brazil (P.1 lineage) with mutations in the S gene are spreadingglobally. To analyze effects on neutralization elicited by BNT162b2, weengineered S mutations from each of the three new lineages intoUSA-WA1/2020, a relatively early isolate of the virus from January 2020(FIG. 138 ). We subsequently produced five recombinant viruses. Thefirst had all the mutations found in the S gene in the B.1.1.7 lineage(B.1.1.7-spike), the second had all the mutations found in the S gene inthe P.1 lineage (P.1-spike), the third had all the mutations found inthe S gene in the B.1.351 lineage (B.1.351-spike), the fourth had anN-terminal domain deletion found in the B.1.351 lineage and the globallydominant D614G substitution (B.1.351−A242−244+D614G), and the fifth hadthe three mutations from the B.1.351 lineage at the receptor-bindingsite (K417N, E484K, and N501Y) and a D614G substitution(B.1.351−RBD+D614G). The amino acid residues mutated in theB.1.351−RBD+D614G virus are also among those mutated in the P.1 lineagevirus, though in the P.1 lineage virus, K417 is mutated to threoninerather than asparagine. All the mutant viruses yielded infectious viraltiters exceeding 10⁷ plaque-forming units per milliliter. TheB.1.1.7-spike and B.1.351-spike virus formed plaques that were smallerthan those of the other viruses (FIG. 139 ).

We performed 50% plaque reduction neutralization testing (PRNT₅₀) using20 serum samples that had been obtained from 15 participants in thepivotal trial (Polack F P et al. Safety and efficacy of the BNT162b2mRNA Covid-19 vaccine. N Engl J Med 2020; 383: 2603-15; Walsh E E et al.Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates.N Engl J Med 2020; 383: 2439-50) 2 or 4 weeks after the administrationof the second dose of 30 μg of BNT162b2 (which occurred 3 weeks afterthe first immunization) (FIG. 140 ). All the serum samples efficientlyneutralized USA-WA1/2020 with almost all of them having titers higherthan 1:40. Geometric mean neutralizing titers against USA-WA1/2020,B.1.1.7-spike, P.1-spike, B.1.351-spike, B.1.351−A242−244+D614G, andB.1.351−RBD+D614G viruses were 532, 663, 437, 194, 485, and 331,respectively (FIG. 141 and Table 31). Thus, as compared withneutralization of USA-WA1/2020, neutralization of B.1.1.7-spike andP.1-spike viruses was roughly equivalent, and neutralization ofB.1.351-spike virus was still robust but ^(˜)2.7-fold lower. Our dataare also consistent with lower neutralization titers against the viruswith the full set of B.1.351-spike mutations than virus with eithersubset of mutations and suggest that mutations in the receptor-bindingsite (K417N, E484K, and N501Y) affect neutralization more than the242-244 deletion in the N-terminal domain of the spike protein.

Because neutralization of the B.1.1.7-spike and P.1-spike viruses byBNT162b2-elicited sera is roughly equivalent to neutralization ofUSA-WA1/2020, the neutralization data provide strong support thatBNT162b2 will continue to protect against the variants first detected inthe UK or Brazil. Protection against B.1.351 lineage virus is alsoanticipated, given that, although neutralization titers against thisvariant are somewhat lower, they are still robust and much higher thanthe barely detectable titers observed after one dose of BNT162b2, whenstrong efficacy was already observed in the pivotal C4591001 efficacytrial (Polack F P et al. Safety and efficacy of the BNT162b2 mRNACovid-19 vaccine. N Engl J Med 2020; 383:2603-15; Walsh E E et al.Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates.N Engl J Med 2020; 383:2439-50; Sahin U et al. BNT162b2 inducesSARS-CoV-2-neutralising antibodies and T cells in humans. Dec. 11, 2020(www.-medrxiv.-org/-content/-10.-1101/-2020.-12.-09.-20245175v1).preprint.). In addition, T cell immunity may also be involved inprotection (Liao M et al. Single-cell landscape of bronchoalveolarimmune cells in patients with COVID-19. Nature Medicine 2020/), andBNT162b2 immunization elicits CD8+ T-cell responses that recognizemultiple variants (Skelly D T et al. Vaccine-induced immunity providesmore robust heterotypic immunity than natural infection to emergingSARS-CoV-2 variants of concern. Research Square 2021).

Materials and Methods Construction of Isogenic Viruses.

All recombinant SARS-CoV-2s with spike mutations (FIG. 138 ) wereprepared on the genetic background of an infectious cDNA clone derivedfrom clinical strain USA-WA1/2020 (Xie X et al. An Infectious cDNA Cloneof SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3). The mutations wereintroduced into the spike gene using a PCR-based mutagenesis protocol asreported previously (Plante J A et al. Spike mutation D614G altersSARS-CoV-2 fitness. Nature 2020. doi: 10.1038/s41586-020-2895-3; Xie Xet al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K andN501Y variants by BNT162b2 vaccine-elicited sera. Nat Med 2021. doi:10.1038/s41591-021-01270-4). The full-length infectious cDNAs wereligated and used as templates to in vitro transcribe full-length viralRNAs. The original viral stocks (P0) were recovered from Vero E6 cellson day 2 post electroporation of the in vitro transcribed RNAs. The P0viruses were propagated on Vero E6 cells for another round to produce P1viruses for the neutralization assays. The titers of P1 viruses weremeasured by plaque assay on Vero E6 cells (FIG. 139 ). The completespike sequences of the P1 viruses were confirmed by Sanger sequencing tohave only the intended nucleotide changes from the USA-WA1/2020sequence. A detailed protocol of the above experiments was recentlyreported (Xie X et al. Engineering SARS-CoV-2 using a reverse geneticsystem. Nature Protocols 2021:doi.org/10.1038/s41596-021-00491-8).

Serum Specimens and Neutralization Assay.

FIG. 140 illustrates the immunization and serum collection scheme. Aconventional 50% plaque-reduction neutralization test (PRNT₅₀) wasperformed to quantify the serum-mediated virus suppression as previouslyreported (Muruato A E et al. A high-throughput neutralizing antibodyassay for COVID-19 diagnosis and vaccine evaluation. Nat Commun 2020;11:4059). Briefly, individual sera were 2-fold serially diluted inculture medium with a starting dilution of 1:40 (dilution range of 1:40to 1:1280). The diluted sera were incubated with 100 PFU of USA-WA1/2020or mutant SARS-CoV-2. After 1 h incubation at 37° C., the serum-virusmixtures were inoculated onto a monolayer of Vero E6 cells pre-seeded on6-well plates on the previous day. A minimal serum dilution thatsuppressed >50% of viral plaques is defined as PRNT₅₀. Theneutralization titers are presented in Table 31.

TABLE 31 PRNT₅₀'s of twenty BNT162b2 post-immunization sera againstUSA-WA1/2020 and mutant SARS-CoV-2. *PRNT₅₀ B.1.351- Δ242- B.1.351-USA-WA1/2020 B.1.1.7- P.1-spike B.1.315-spike 244 + RBD + Serum ID Exp1Exp2 Exp3 GMT spike Exp1 Exp2 GMT Exp1 Exp2 GMT D614G D614G 1 320 320320 320 640 320 320 320 160 160 160 320 320 2 160 160 160 160 160 80 8080 40 40 40 160 80 3 640 640 640 640 640 640 640 640 320 320 320 640 6404 160 320 320 254 320 320 320 320 80 160 113 160 80 5 320 320 320 320640 320 320 320 160 160 160 320 320 6 320 640 640 508 320 640 320 453160 160 160 320 160 7 1280 640 640 806 1280 640 1280 905 320 320 3201280 1280 8 320 320 320 320 320 160 160 160 80 80 80 160 160 9 1280 12801280 1280 1280 1280 640 905 640 640 640 1280 1280 10 640 640 1280 8061280 640 320 453 640 320 453 1280 640 11 320 320 320 320 640 320 320 32080 160 113 320 160 12 640 640 640 640 640 640 320 453 160 160 160 320320 13 1280 1280 1280 1280 1280 640 640 640 160 320 226 1280 640 14 320320 640 403 320 320 160 226 160 80 113 320 160 15 640 640 640 640 6401280 640 905 320 320 320 640 320 16 320 320 640 403 1280 640 320 453 160320 226 640 320 17 1280 1280 1280 1280 1280 1280 640 905 320 320 3201280 640 18 640 640 640 640 640 640 320 453 160 320 226 320 320 19 640640 640 640 640 1280 640 905 320 320 320 640 640 20 640 640 640 640 1280640 320 453 160 160 160 640 320 ^(†)GMT 502 520 577 532 663 520 368 437184 204 194 485 331 ^(#)95% CI 371- 401- 443- 409- 497- 372- 275- 325-133- 151- 144- 345- 228- 680 674 751 693 884 726 491 589 255 276 261 681480 *The data for USA-WA1/2020 are from three experiments; the data forB.1.1.7-spike, B.1.351-Δ242-244 + D614G, and B.1.351-RBD-D614G virusesare from one experiment each; and the data for P.1-spike andB.1.351-spike viruses are from two experiments. For each independentexperiment, individual PRNT50 value is the geometric mean of duplicateplaque assay results; no differences were observed between the duplicateassays. ^(†)Geometric mean neutralizing titers. ^(#)95% confidenceinterval (95% CI) for the GMT.

Example 39: Durability of BNT162b2-Induced CD4+ and CD8+ T-CellResponses

In a subset of 24 subjects across dose levels 10 to 30 μg, samplescollected at Day 85 and Day 184 (nine and 23 weeks post-boost,respectively) were analyzed in order to determine the durability ofT-cell responses induced by BNT162b2. On Day 184 and after an initialcontraction, both CD4⁺ and CD8⁺ T-cell responses were detectable in themajority of individuals, across the three dose levels tested. Kineticsof CD4⁺ and CD8⁺ responses observed in four older adult subjectsvaccinated with 10 μg BNT162b2 were comparable to younger adultsubjects, with S protein-specific CD4⁺ T cells still detectable in allfour subjects 23 weeks after boost vaccination. BNT162b2 induced CD4⁺and CD8⁺ responses were either higher than or in the range of recallantigen memory responses (Error! Reference source not found.42).

Example 40: MHC-I Binding Epitopes Recognized by CD8 T-Cells Induced byBNT162b2

Using MHC-class I multimer technology, several epitopes spread acrossthe whole length of the S Protein and presented by a combination ofcommon HLA-A and HLA-B alleles were identified to be recognized by CD8+T-cells induced by BNT162-b2 (measured 7 days after the boostvaccination). Some peptide/HLA combinations were found in more than onesubject.

TABLE 32 MHC-I binding epitopes recognized by CD8 T-cells induced byBNT162b2 Position in Identified in HLA Epitope SEQ ID NO S Protein No.Subjects B35:01 LPFNDGVYF 47 84-92 1 A03:01 GVYFASTEK 52 89-97 1 A02:01YLQPRTFLL 40 269-277 3 B35:01 QPTESIVRF 45 321-329 1 A26:01 CVADYSVLY 53361-369 1 B15:01 CVADYSVLY 53 361-369 1 A03:01 KCYGVSPTK 54 378-386 2A24:02 NYNYLYRLF 43 448-456 3 B15:01 FQPTNGVGY 55 497-505 1 B35:01IPFAMQMAY 46 896-904 1 A02:01 RLQSLQTYV 41 1000-1008 2 A68:01 GTHWFVTQR56 1099-1108 1 C04:01 VYDPLQPEL 57 1137-1145 1 A24:02 QYIKWPWYI 421208-1216 3 A24:02 KWPWYIWLGF 44 1211-1220 1

Example 41: Histological Findings Following Administration of BNT162b2

Classical chromogenic immunohistochemistry (IHC) and chromogenic dualIHC-ISH (in situ hybridization) experiments were performed toinvestigate biodistribution of BNT162b2 in mouse tissues 6h and 6dpost-injection.

Protocol

After harvesting, tissue is fixed in 4% RotiHistofix overnight at 4° C.and embedded in paraffin wax after dehydration in Leica TissueProcessor. Chromogenic IHC is performed. Spike protein is detected withanti-Spike2 mouse monoclonal antibody (Genetex). Dual IHC-ISH assay isconducted according to a self-established protocol based on Document MK51-149 from Advanced Cell Diagnostics using the company kits andreagents. BNT162b2 probe (modV9) for ISH is custom designed by AdvancedCell Diagnostics based on the sequence provided by TRON. IHC protocolfor the immune cell markers CD11c (Cell Signaling), CD19 (CellSignaling), CD169 (Thermo Fisher) and F4/80 (Cell Signaling) were inplace at TRON and they are adapted to dual IHC-ISH assay for theproject. Images are acquired using Vectra Polaris Multispectral SlideScanner microscope (Akoya Bioscience) and analysed with PhenoChartsoftware (Akoya Bioscience).

Results

As can be seen in FIG. 143 , a specific vaccine mRNA signal (red) isdetected in the lymph nodes (LN) 6h post injection using modV9 probe indual IHC-ISH assay. Vaccine is mostly localized to subcapsular sinus (LNin 9 and 5 positions) and B cell follicles (LN in 12 and 1 positions).Dendritic cells are visualized by CD11c staining (turquoise, upperimages) and only some of them uptake the vaccine. Majority of CD169+macrophages (subcapsular sinus macrophages, turquoise, middle images)are positive for the vaccine. B cells (CD19+, turquoise, lower images)are the second major population showing vaccine signal.

A specific vaccine mRNA signal is still detectable in the LN 6d postinjection using modV9 probe in dual IHC-ISH assay, albeit in very lessamount (data not shown). Some CD11c+ DCs and subcapsular sinusmacrophages are positive for the vaccine. Most of the vaccine signaldetected is in the B cells (CD19+).

As can be seen in FIG. 144 , a specific vaccine mRNA signal (red) isdetected in the spleen 6h post injection using modV9 probe in dualIHC-ISH assay. Majority of the vaccine signal is detected in the whitepulp. Dendritic cells are visualized by CD11c staining (turquoise, upperimages) and only some of them uptake the vaccine. A small portion ofF4/80+ macrophages (turquoise, middle images) uptake the vaccine. Bcells (CD19+, turquoise, lower images) are the major population showingthe vaccine signal.

A specific vaccine mRNA signal is still detectable in the spleen 6d postinjection using modV9 probe in dual IHC-ISH assay, albeit in very lessamount (data not shown). The vaccine signal detected is solely in the Bcells (CD19+). No DCs and macrophages show vaccine signal 6dpost-injection.

After 6h, using mouse anti-S2 mouse monoclonal antibody, we detected asignal in the muscle, especially in some muscle fibers and in theconnective tissue perimysium. In the LNs, we detected cells expressingSpike protein in the T cell zone (data not shown).

After 6d, using mouse anti-S2 mouse monoclonal antibody, no Spikeexpression is detectable in the muscle. On the contrary, LNs are full ofcells expressing the vaccine (data not shown).

No nonspecific staining is detected with the S2 mouse monoclonalantibody in the chromogenic IHC experiments.

Summary

A very strong vaccine signal is visible in the draining LNs and spleen6h post-injection. In the LN, vaccine is mostly detected in B cellfollicles and subcapsular sinus, with some signal also in the T cellzone. By dual IHC-ISH, we showed that indeed the B cells (CD19+) andsubcapsular sinus macrophages (CD169+) are the major cells that uptakethe vaccine. Dendritic cells (CD11c+) in the T cell zone andintermediary sinus also uptake the vaccine. After 6d, some vaccine mRNAis still visible in the draining LNs. The signal observed in the T-cellzone after 6d is in the dendritic cells (CD11c+). Some B cells and LNmacrophages also still have some vaccine at that stage.

Analysis of the spleen harvested 6h post-injection showed that vaccinealready reaches spleen within 6h, most probably via blood circulation.The signal is located white pulp, where B cells and T cells form themajor population and antigen presentation to T cells occur in the whitepulp. With dual IHC-ISH assay, we showed that majority of B cells uptakethe vaccine. Many DCs (CD11c+) surrounding the B cells are alsopositive. After 6d, signal is restricted to B cells. IHC protocol isestablished to detect spike protein expression using anti-Spike S2 mousemonoclonal antibody on the cell pellets treated with/without thevaccine. A specific signal is detected only in the cells treated withBNT162b2. No nonspecific staining was visible in the naïve tissuestested. In the muscle, Spike expression is detected 6h post injection inthe muscle fibers and in the connective tissue perimysium. After 6d, nostaining is detectable in the muscle. On the contrary, the massive Spikeexpression is visible 6d post injection in LN, in particular in theT-cell zone.

Example 42: Stability Studies

Stability assessments of BNT162b2 formulations at various concentrations(e.g., 0.5 mg/mL, 1 mg/mL, and 2 mg/mL) have been performed, and haveincluded assessments of compositions, stored at various temperatures(e.g., −70° C. [e.g., −70±10° C.], −20° C. [e.g., −20±5° C.], +5° C.[e.g., 5±3° C.], or +25° C. [e.g., 25±2° C.]) and/or for various periodsof time (e.g., 0.5 months, 1 month, 2 months, 3 months, 4 months, and incertain cases one or more intervening time points (e.g., 1.5 months, 2.5months, etc).

In exemplary studies, mice were injected (single leg) at day 0 with 20uL of a relevant formulation. Blood was collected and serum generated atdays 14, 21, and 28 after the administration; spleen were isolated atday 28.

ELISAs were performed to detect presence of antibodies in serum thatbind to S1 protein, or specifically to the RBD domain. FIG. 145 presentsexemplary S1 ELISA results obtained with 28-day serum from mice injectedwith indicated formulations that had been stored under indicatedtemperature conditions for indicated periods of time. As can be seenwith reference to FIG. 145 , all stored samples performed well, andreasonably comparably, after one (1) month of storage. After two (2) orthree (3) months of storage, some decreased activity was observed forsamples stored at +25° C., but samples stored at −70° C., −20° C., +5°C. maintained significant performance.

At some time points, one or more parameters such as appearance, RNAcontent, RNA integrity, RNA encapsulation, lipid content (overall and/orof individual components and/or ratios thereof), particle size, particlepolydispersity index, in vitro expressability, etc) were assessed;additional or alternative parameters may be or have been assessed.

Exemplary observations include that storage at +25° C. is notrecommended for periods of time longer than about 2 weeks, andpreferably not more than about 1 week as, among other things, RNAintegrity was observed to decrease significantly. It was also observedthat, in at least some cases, significant ability to induce antibodieswas maintained even when in vitro expressability was materially reduced.Change in polydispersity index, particularly after about 3 months ofstorage, or after about 4 months of storage, were observed to be greaterfor formulations stored at +5° C. or above than for those stored atlower temperatures.

1.-73. (canceled)
 74. A pharmaceutical composition comprising: (a) anRNA comprising a nucleotide sequence that includes modified uridines andencodes the polypeptide of SEQ ID NO: 7, which nucleotide sequence is atleast 90% identical to SEQ ID NO: 9; formulated in (b) lipidnanoparticles comprising each of: (i) a cationically ionizable lipid;(ii) a sterol; (iii) a neutral lipid; and (iv) a lipid conjugate,wherein the cationically ionizable lipid is or comprises((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),the sterol is or comprises cholesterol, the neutral lipid is orcomprises a phospholipid, and the lipid conjugate is or comprises apolyethylene glycol (PEG)-lipid.
 75. The pharmaceutical composition ofclaim 74, wherein the phospholipid is or comprisesdistearoylphosphatidylcholine (DSPC).
 76. The pharmaceutical compositionof claim 74, wherein the PEG-lipid is or comprises 2-[(polyethyleneglycol)-2000]-N,N-ditetradecylacetamide.
 77. The pharmaceuticalcomposition of claim 74, wherein the lipid nanoparticles comprise eachof: (a)((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate);(b) cholesterol; (c) distearoylphosphatidylcholine (DSPC); and (d)2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
 78. Thepharmaceutical composition of claim 74, wherein the RNA comprises a5′-cap that is or comprises m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG.
 79. Thepharmaceutical composition of claim 74, wherein the RNA comprises apolyA sequence, wherein the polyA sequence comprises 30 adeninenucleotides followed by 70 adenine nucleotides, wherein the 30 adeninenucleotides and 70 adenine nucleotides are separated by a linkersequence.
 80. The pharmaceutical composition of claim 74, wherein theRNA comprises a 5′-UTR that is or comprises a modified humanalpha-globin 5′-UTR.
 81. The pharmaceutical composition of claim 74,wherein the RNA comprises a 3′-UTR that is or comprises a first sequencefrom the amino terminal enhancer of split (AES) messenger RNA and asecond sequence from the mitochondrial encoded 12S ribosomal RNA. 82.The pharmaceutical composition of claim 74, wherein the cationicallyionizable lipid is within a range of about 40 to about 50 mole percent,the sterol is within a range of about 35 to about 45 mole percent, theneutral lipid is within a range of about 5 to about 15 mole percent, andthe lipid conjugate is within a range of about 1 to about 10 molepercent.
 83. The pharmaceutical composition of claim 77, wherein((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) iswithin a range of about 40 to about 50 mole percent, cholesterol iswithin a range of about 35 to about 45 mole percent,distearoylphosphatidylcholine (DSPC) is within a range of about 5 toabout 15 mole percent, and 2-[(polyethyleneglycol)-2000]-N,N-ditetradecylacetamide is within a range of about 1 toabout 10 mole percent.
 84. The pharmaceutical composition of claim 83,which is in a liquid formulation.
 85. The pharmaceutical composition ofclaim 83, which is in a frozen formulation.
 86. The pharmaceuticalcomposition of claim 74, wherein the formulation comprises an aqueouscryoprotectant buffer.
 87. The pharmaceutical composition of claim 74,wherein the RNA has a structure represented by m₂ ^(7′3′-O)Gppp(m₁^(2′-O))ApG-hAg-Kozak-S1S2-PP-FI-A30L70.
 88. The pharmaceuticalcomposition of claim 87, wherein the RNA comprises the nucleotidesequence of SEQ ID NO:
 20. 89. The pharmaceutical composition of claim88, wherein the nucleotide sequence includes modified uridines in placeof all uridines.
 90. The pharmaceutical composition of claim 89, whereinthe modified uridines are each N1-methyl-pseudouridine.
 91. Thepharmaceutical composition of claim 74, wherein the nucleotide sequencethat includes the modified uridines and encodes the polypeptide of SEQID NO: 7 is codon-optimized for human subjects.
 92. The pharmaceuticalcomposition of claim 74, wherein the nucleotide sequence that includesthe modified uridines and encodes the polypeptide of SEQ ID NO: 7 ischaracterized in that its G/C content is increased by about 55% ascompared to the G/C content of the nucleotide sequence of SEQ ID NO: 2.93. A method of vaccinating by administering a pharmaceuticalcomposition comprising: (a) an RNA comprising a nucleotide sequence thatincludes modified uridines and encodes the polypeptide of SEQ ID NO: 7,which nucleotide sequence is at least 90% identical to SEQ ID NO: 9;formulated in (b) lipid nanoparticles comprising each of: (i) acationically ionizable lipid; (ii) a sterol; (iii) a neutral lipid; and(iv) a lipid conjugate, wherein the cationically ionizable lipid is orcomprises((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),the sterol is or comprises cholesterol, the neutral lipid is orcomprises a phospholipid, and the lipid conjugate is or comprises apolyethylene glycol (PEG)-lipid.
 94. The method of claim 93, wherein thestep of administering comprises administering more than one dose of thepharmaceutical composition.
 95. The method of claim 94, wherein the morethan one dose comprises a first dose and at least a second dose, andwherein the first and second doses are spaced apart by a time period ofabout 21 days.
 96. The method of claim 93, wherein the step ofadministering comprises administering at least one dose eachindependently comprising RNA in an amount within a range of about 1 μgto about 100 μg.
 97. The method of claim 95, wherein the step ofadministering comprises administering at least two doses, each of whichcomprises about 30 μg of the RNA.
 98. The method of claim 93, whereinthe RNA comprises the nucleotide sequence of SEQ ID NO:
 20. 99. Themethod of claim 98, wherein the nucleotide sequence includes modifieduridines in place of all uridines.
 100. The method of claim 99, whereinthe modified uridines are each N1-methyl-pseudouridine.
 101. A method ofmanufacturing the pharmaceutical composition of claim 74, comprisingcombining the RNA with the cationically ionizable lipid, the sterol, theneutral lipid, and the lipid conjugate to form lipid nanoparticles thatencapsulate the RNA.
 102. The method of claim 101, wherein the RNAcomprises the nucleotide sequence of SEQ ID NO:
 20. 103. The method ofclaim 102, wherein the nucleotide sequence includes modified uridines inplace of all uridines.